Relationship between Secretion of Platelet
Factor 4 and Thrombin Generation during In
Vitro Blood Clotting
Marc A. Shuman
J Clin Invest. 1980;65(2):307-313. https://doi.org/10.1172/JCI109673.
We have studied the effects of both impaired prothrombin activation and direct inhibition of thrombin on the platelet release reaction in clotting blood to determine the role of thrombin in this process. In blood from two patients with congenital Factor V deficiency, prothrombin activation during spontaneous in vitro clotting was delayed and decreased. Secretion of platelet Factor 4 was also delayed and was detected only after thrombin formation was initiated. Addition of a small amount of normal plasma to the patients' blood in vitro
corrected the abnormalities in both thrombin formation and the platelet release reaction in parallel fashion. A delay in the onset of secretion of platelet Factor 4 was also observed when thrombin generated in normal blood during spontaneous in vitro clotting was inhibited by either purified hirudin or anti-thrombin Fab. These observations suggest that thrombin is the essential stimulus for platelet secretion during in vitro blood clotting.
The effect of inhibitors of the platelet release reaction on prothrombin activation during in vitro blood clotting was also studied. When either prostacyclin or the combination of
prostaglandin E1 and N6O2¢-dibutyryl cyclic AMP was added, secretion of platelet Factor 4 was inhibited 85-95%. We were unable to detect any inhibition of initiation of prothrombin activation or inhibition of that part of thrombin generation associated with clotting. These results indicate either […]
Research Article
Find the latest version:
Relationship between Secretion of Platelet
Factor 4 and
Thrombin Generation during
In
Vitro
Blood
Clotting
MARC A.SHUMAN,Division ofHematology-Oncology, Department of Medicine, University of California, San Francisco, California 94143
SHIRLEY P.LEVINE,Audie L. Murphy Memorial Veterans Administration Hospital and the Division of Hematology, Department ofMedicine, University of Texas Health ScienceCenter, SanAntonio, Texas 78284
A BST R A C T We have studied the effects of both impaired prothrombin activation and direct inhibition of thrombin onthe
platelet
releasereactioninclotting bloodtodetermine the role of thrombininthis process. In blood from two patients with congenital FactorVdeficiency,prothrombinactivationduring spontaneous in vitroclottingwas delayed and decreased. Secretion of plateletFactor4wasalsodelayedand was detected only after thrombin formationwasinitiated. Addition of asmall amountof normal plasma to the patients' blood in vitro corrected the abnormalities in both thrombin formation and the
platelet
release reaction inparallel fashion. Adelay
in the onset ofsecretion ofplatelet
Factor4was also observed when
thrombin
generated in normal blood during spontaneous in vitro clotting wasinhibited by either purified hirudin or anti-throm-bin Fab. These observations suggest that thrombin is*the
essential stimulus for platelet secretion during in vitro blood clotting.The effect of inhibitors of the platelet release reac-tion on prothrombin activation during in vitro blood clotting was also studied. When either prostacyclin or the combination of prostaglandin E1 and N6021-dibutyryl cyclicAMP wasadded, secretion of platelet Factor 4 was inhibited 85-95%. We were unable to detect any inhibition of initiation of prothrombin activation or inhibition of that part of thrombin gen-erationassociated with clotting. These results indicate either that the platelet release reaction may not be required for the initiation of prothrombin activation or
only
a very limited amount of secretion may be necessary for normal generation of thrombin to occur.Apreliminaryreportofthiswork was presented at the An-nual Meetingofthe AmericanFederation forClinical Research, Washington, D.C., May 1979, and was published in abstract forn in 1979.Clin. Res. 27: 307A.
Dr. Levine is aClinicalInvestigator.
Receivedfor publication6August 1979andinrevisedform 28 September1979.
INTRODUCTION
The interactionbetween platelets andclottingfactors is a complex one. Activation of the coagulation sys-tem results in the formation ofthrombin, which clots fibrinogen, andinaddition stimulates both the
platelet
releasereaction'andplatelet aggregation. Platelets,on the other hand, serve as cofactors in two reactions in
thecoagulation pathway: theactivationofFactorXand the activation of
prothrombin
(1). Evidence ofa role forplateletsinthe early steps ofthe contact activation pathway has also been presented (2). Using radio-immunoassays for thrombin anda protein secretedby platelets, platelet Factor4 (PF4),2 we havepreviously
shown thataclosecorrelation existsbetween thrombin formation and the
platelet
release reactionduring
spontaneous invitroclotting of normal blood (3). The onset of thrombin formation and secretion of PF4 precedes visible clotting by severalminutes.The con-centrations ofthrombin and PF4 in plasma then rise gradually in a parallel fashion until shortly before clotting, when, atrelatively high thrombin concentra-tions, the major portion of platelet secretion of PF4 occurs.
We now report the results of experiments that further clarify the relationship between prothrombin activation and theplatelet releasereaction. Inthe first series of experiments, we have examined the conse-quences ofdelayed prothrombin activation on initia-tionof theplatelet release reaction during the clotting ofblood from two women with congenital Factor V deficiency. To determine the effects on the platelet release reaction of direct inhibition of the thrombin formed during spontaneous clotting, we added either
IThe term "release reaction" refers to platelet secretion
and the terms are used interchangeably.
2Abbreviations used in this paper: dbcAMP, N602'-dibu-tyryl cyclic AMP;PGE,, prostaglandin El;PGI2,prostacyclin; PF4,platelet Factor 4.
hirudin or ananti-thrombin antibody tofreshly drawn normail l)lood and measured the subsequent platelet secretion ofPF4.
In a second series of experiments, to blood from
normalvolunteers weaddedagentsthat elevate plate-let cyclicAMPand measured subsequent thrombin and
PF4 formation. Thepurpose of these experiments was to determine how inhibition of platelet secretion af-fects the initiation and time-course of thrombin
generation during spontaneous clotting.
XIETHODS
Materials. Sources for most reagents have been reported (3-5). Prostacyclin(PGI2) wassynthesized by K. C. Nicolau of the University of Pennsylvania, Philadelphia, Pa. (6) andkindly suppliedby Dr. J. Bryan Smith of Thomas Jeffer-son Medical College, Philadelphia, Pa. Rabbit anti-human thrombin Fab and Fab from normal rabbit immunoglobulin (Ig)G were prepared as described (7). Prostaglandin El
(PGE,) was generously provided by Ono Pharmaceutical Co., Ltd., Japan.
Very high purity hirudin (10,000-12,000 U/mg) was generously provided by Dr. J. Fenton, II, Albany, N. Y.
The original material had been purified by Dr. F. Mark-wardt, East Germany, and recycled by Dr. Fenton after crystallographic studies of hirudin and its complex with thrombin.3 One hirudin unit is defined as that which inhibitsone NIH unitof thrombin.
Thromvbin generation and secretion of PF4. With the two syringe technique, blood was obtained from the two patients and from normal volunteers and was immediately placedinto newpolypropylenetubes. One of the tubes con-tained noother additions and the bloodwas allowed toclot
spontaneouisly. Asecond tube contained3.8%sodium citrate (1:9, vol/vol) and was used as an anticoaguilated control (3). Additional tubes contained substances that were added to the blood to determine their effect on prothrombin activation and plateletsecretion (see below). Samples were removed from each tube immediately after collection of the blood and at regular intervals, and were assayed for either PF4 orthrombin(3-5).
PF4values ofsamplesfrom thetube containing anticoagu-latedblood were subtracted from PF4 values of blood meas-ured at comparable times after collection in the tubes not containing anticoagulant (3). The net basal values ranged between 0-50 ng/ml depending upon the interval elapsed from venipuncture to processingof the first sample.
Studies in patients with congenitalFactor V deficiency.
Two patients with severecongenital FactorVdeficiencywere studied: patient K.H. on one occasion and patient E.N. on two occasions. Blood was drawn and distributed into three tubes as described above. To the third tube, fresh normal plasma was added as asourceof FactorV.
Effects of hirudin. Blood was obtained from normal donors and distributed intothree tubes as described above. Hirudin wasimmediately added to the third tubein afinal concentration of5U/ml. This experimentwas performedon
bloodfrom twovolunteers.
Effects ofatti-thrombin Fab. Bloodobtained from normal donors was distributed into four tubes as described above. Anti-thrombin Fab was added tothe third tube and control
'Landis,B. H., J.W.Fenton, II, andF. Markwardt.
Manu-scriptin preparation.
rabbit Fab to a fourth, each at a final concentration of 0.16 mg/ml. Two donors were used for this experiment.
Effect of antiplatelet agents. (a) Blood was collected from normal volunteers and distributed into three tubes. To the third, PGI2 vasadded ina final concentrationi of50 nM.
Fouir donors were used for thisexperimlenit.
(b) PGE, and N602'-dibutyryl cyclic AMP (dbcAMP) were added to freshly- drawn blood in a finial concenitrattioni of 10,ug/mland 1 mM\, respectivelv. ThePGE,stock prepara-tion w\as dissolved in etlhainol at a concenitraprepara-tioni of 10 mg/mii. The dbcAMP preparation (100 mM)was dissolved in 0.14 MI NaCl-0.01 M sodiumln phosphate, pH 7.4. Two donors were used in this experiment.
(c) Three normalvolunteers ingested 600 mg of aspirini It
12-h intervals beginning 24 h before blood collection (total dose, 1,800 mg). Serum salicylate levels were measured to assess compliance (8). Bloodwas collectedfrom each xoltin-teer and thrombin formation and secretion of PF4 deter-mined asdescribed above.
RESULTS
Prothrombin activation and the platelet release reaction in FactorV-deficient
patients.
Todetermine the effect ofdelayedprothrombinactivation on platelet secretion ofPF4, bloodwas obtained fromtwopatienits with congenital FactorVdeficiency (plasma Factor \V, 0%) (9). Immediately after bloodl collection, serial samples were removed for assay ofPF,
and thrombin in plasma and serum. The onset of tlhrom)bin formal-tion was markedly delayed in the blood from patient E.N., a woman with a severe bleeding history (Fig. IA). Nothrombin was detectable for over 100 min after bloodcollection (normal,4-8min),and once initiated, thrombin generation progressed slowly. The interval from initiation of thrombin formation to clotting, which occurred at203 min, was prolonged (patient,46 mim; normal, 12-24 min). The concentration of thromnbin in serum was reduced (patient, 80 ng/ml; normal,500-1,500 ng/ml).
An abnormality was also seen in the platelet re-lease reaction. The plasma PF4 concentrations in the clotting samples were no higher than in the anti-coagulated control samples for the first 200 min. Be-causeonly alimitedamountof blood remained inthe tube, we were unable to measure PF4 levels during the time of maximal thrombin formation (206-240 min); however, the PF4 concentration in an
over-nightserum specimen was normllal (5,900ng/lmll). When thepatient's bloodwasmixeclwith 1/18(vol/vol) fresh normalplasma(Fig IA,P+ C), the abnormalities described above were almost completely corrected (clottingoccurredat 27min).Theserumthrombin
coni-centration wasonly slightlydecreased(400
ng/ml),
ainda comparable improvement in the plateletrelease re-action was observed. SecretionofPF4wasinitiatedby
18 min and increased rapidly over the next 18 min, parallelingthrombinformation. This latter observation indicates that the markeddelayintheplateletsecretion
of the blood of patient E.N. described above was not
1000 800 600 400
40 80 120 160 200
TIME(minutes)
I
°
Ir
-200C
100(
I
800 600
P -400
200
p
i 100
240 16h
E
C~
0
I-w
uJ
a--E
z
z
I-I
201
10
-I
I~ I
dI
40 80 120 160 200 240 TIME(minutes)
FIGURE 1 Thrombin generation and secretion ofPF4 in blood frompatients with congenital
FactorVdeficiency. Bloodwasdrawn from patients(patient E.N., Fig.1A;patient K.H., Fig. IB),
immediatelyplacedintopolypropylene tubes, andallowedtoclotspontaneously.Toonetube, freshnormalplasmawasaddedimmediatelyaftercollection of the blood.Sampleswereremoved
serially and assayed for thrombin (0, 0) orPF4(A, A). P+C, mixture ofpatient's bloodand
normalplasma(closed symbols); P, Patient'sblood (open symbols).
because ofanabnormalityinthe release reaction,but
rathertoan inadequate thrombin stimulus.
The secondpatient(patient K.H.) hadamuch milder
clinicalhistory of bleedingproblems. Thrombin
forma-tion was abnormal in thepatient's blood (Fig. 1B) but
these abnormalities were less than those observed in
the firstpatient.Thrombinwasfirstdetectedat48min,
and the interval until clotting was only slightly
pro-longed,28 min(clottingtime, 76min). The final throm-binconcentration (200 ng/ml), although reduced, was
2.5-fold higher than that ofthe first patient's serum.
The onsetof PF4secretion wasalsodelayed (50 min),
but in contrast to the first patient, virtually complete plateletsecretionhadoccurred by thetimeof clotting. The delay in the release reaction was similar to the delayin prothrombin activation,andPF4 secretion oc-curred only after thrombin formation was initiated. When thispatient's bloodwasmixed 1/10 (vol/vol) with
normalplasma (Fig. 1B), the abnormalitiesinthrombin
formation and platelet secretion were corrected in a
parallel manner(clotting time, 31 min).
These studiessuggestthatthrombinformation must
occurbeforeplateletsecretioncantakeplace, and that athresholdconcentrationof thrombinmustbe reached before release of PF4 is initiated. Furthermore, the
addition of normalplasmato correctFactorV-deficient blood resultsinasimultaneous correctionof thrombin
generation andplateletsecretion.
Effect of inhibition of thrombinactivityonplatelet release ofPF4. Toexamine further the role of throm-bin in platelet secretion during spontaneous in vitro
blood clotting, specific inhibitors of thrombin were
addedtonormal blood immediatelyafteritscollection, and therelease of PF4wasdeterminedinserial samples.
Additionof the leech anti-thrombin, hirudin, toblood
resultedin an inhibition of clotting (hirudin, 38 min;
control,24min)andplateletsecretion.Both the time of onsetofplateletsecretion ofPF4andcompletion were
delayed (Fig. 2).Wewereunabletomeasurethrombin
1-0 C
0
-u
IL
20,000
10,000
8000 6000 4000
2000
1000
800 600 400
200
100
/
10 20 30 40 50 TIME(minutes)
FIGURE 2 Effect ofpurified hirudinonsecretionof PF4during clotting of normal blood. Aftervenipuncture,bloodwas
dis-tributed into polypropylenetubes andallowed toclot
spon-taneously. To one tube, purified hirudin was added
im-mediately after collection of the blood. Plasma andserumPF4
concentrations were determined on serial samples from the
control and hirudin-treated blood. (0) Hirudin (5 u/ml); (0) control.
200 100 80
60
40
C
0
z
I-8000
6000
4000
2000 1000 800 600 400
200
100 E
0
I-c
.4:u
uj
U-
!-
I-'4:
accurately by radioimmunoassay in the samples to whichhirudin was added for reasons that are unclear; however, it may be that the 1251-thrombin used in the radioimmunoassay preferentially bound to hirudin rather than to the antibody.
When monospecific Fab fragments of a rabbit anti-bodyagainstthrombinwereaddedtoblood, a prolonga-tionofthe time until clotting was observed (anti-throm-bin Fab,43min; control, 21 min). A delay in the onset of PF4 secretion and a two-fold prolongation of the time at which half-maximal release of PF4 occurred were noted (Fig. 3). Addition of nonimmune rabbit Fabhad noeffectonplateletrelease ofPF4.Thesimilar inhibitory effect onplatelet release of PF4 by two dif-ferent but specific thrombinantagonists wasadditional evidence that thrombin is the primary stimulus for plateletsecretion during blood clotting.
Effect of antiplatelet agents on thrombinformation and the platelet release reaction associated with in vitro blood clotting. To determine whether inhibi-tionofthe platelet releasereactionaffectsprothrombin activation, either PGI2 or
PGE,
and dbcAMP were10,000 _ 8000
6000
-4000
-E 2000
0)
0
P 1000
_
< 800 UL.
_j 600
,> 400_
200-100 1 1 I I
7 14 21 28 35 42 49 TIME (minutes)
FIGURE 3 Effect of anti-thrombin Fab on secretion ofPF4 during clotting of normal blood. Blood was collected,
im-mediately placed into polypropylene tubes,and allowed to
clot.To one tube,anti-thrombin Fab wasaddedafter blood collection; to another, nonimmune Fab. Plasmaand serum
PF4 concentrations were determined on serial samples from each tube.(0) Control; (-)rabbit anti-human thrombin Fab; (A)nonimmune Fab.
added to freshly drawn blood immediately after col-lection. When PGI2 was added to whole blood, the clotting time in four experiments was within the upper limits of normal (clotting time, 27-29 min; normal range, 20-30 min). There was neithera delay in the initial appearanceof thrombin nor a decrease in subse-quent thrombin formation compared to the control samples (Fig. 4). Inhibition (85-95%) of PF4 secretion was observedat 20 min, as shown inFig. 4. Although the thrombin concentration had risen to30ng/ml,only 1.3% ofPF4 release had occurred. Note that 90% of prothrombin activation occurs after clotting of
fibrino-gen (4). Because we were unable to block platelet
secretion of PF4 at the high thrombin concentration present in postclotting serum, we did not obtain
in-formation on the importance ofthe release reaction atthisstage ofclotting.
Similarly, formation of thrombin was not inhibited when
PGE,
and dbcAMP were used to block platelet release of PF4. In fact, a slight shortening of the clotting time (17-20 min) occurred with theaddition of these agents. Initiation of thrombin formation wasprompt and thrombin generation was rapid (Fig. 5). PF4 secretion was inhibited >95% atthe time of
clot-ting. A slight shortening of the clotting time ofwhole blood was also seen with each of these platelet in-hibitingagentswhen used separately (datanotshown). The explanation for this is unclear, but presumably cellular changes were responsible for decreased clot-ting time, sincetheseagentshadnoeffectonthe clot-ting of contact-activated cell-free plasma in which phospholipidwas substituted for platelets.
The results of these experiments indicate either that
the platelet release reaction may not be required for
the initiation of prothrombin activation oronlya very
100 10,000
80_ _8000
60 -6000 -E
E -4000
0)~~~~~~~O
. 20 -2000
z0
X10- ca o / / - 100°0°i
8 /00-8 3
0 6
4
-400
5 10 15 20 25 TIME (minutes)
I i 100
30 35
FIGURE4 Effect ofPGI2 on thrombin formation and secre-tionofPF4duringclottingof normal blood. Bloodwasdrawn andimmediatelydistributedintopolypropylenetubes.To one
tube, PGI2 was added in a final concentration of50 nM.
Samples were removed from each tube and the plasma or
serum assayed forthrombin (0, 0) of PF4 (A, A). Clotting
occurredat 29 min inthe sampletowhich PGI2 was added (O, A)andat 30mininthe control tube(0, A).
0)
E
c
z
a
0
w
I-E
0) C
^
EF. CD
O
I-I
0
5 10 15 20
TIME (minutes)
25
FIGURE 5 Effect ofPGE, and dbcAMPon thrombin forma-tion and secretion of PF4 during clotting of normal blood.
PGE1(final concentration, 10,ug/ml) anddbcAMP(final con-centration 1 mM) were added to blood immediately after
collection. Serial samples were removed and assayed for thrombin(0)orPF4 (A).Clottingoccurredat 20 min.
limitedamountofsecretion maybenecessary for nor-mal generation of thrombin to occur.
Effectof aspirinon the
platelet
releasereaction as-sociated with in vitro bloodclotting.
A variable ef-fect ofprostaglandin synthetase
inhibitors on throm-bin-induced platelet secretion has been reported (10-12). The extent to which platelet secretion isin-hibited is related to both the thrombin concentration and the experimental conditions (e.g., stirring ofthe platelets increases the degree of
inhibition)
(12). Weexamined the effectofaspirinonplateletsecretion dur-ing whole blood clotting, a model that more closely approximates in vivo conditions. Normal donors
in-gested aspirin in an amount sufficient to
completely
inhibit prostaglandin formation(13). Bloodwasdrawn and
assayed
for thrombin andPF4asdescribed for the previous experiments. Initiationof the releasereaction wasnormal, occurringafew minutesafterinitiation of thrombinformation (Fig. 6).Secretionof PF4 proceeded normally andwas essentially complete atthe time of clotting. The serumPF4 concentration, 16.7,g/ml,waswithin the normal range.Thrombin formationwas nor-mal in all three subjects. Because there was no delay in the initiation of the release of PF4 and no dis-sociationbetween thrombin formation and platelet se-cretion of PF4, it appears that even at low thrombin concentrations aspirin does notinhibit the release of alpha granulecontents during invitro blood clotting.
DISCUSSION
Ofthose coagulation factors that have been studied, thrombin alone is capable of directly inducing the platelet releasereaction.Althoughit is known to stim-ulate the release reaction in washed platelets at phys-iologicalconcentrations (4), no prior studies have
dem-E co
C
0
I-L)
1--
I--5 10 15 20 25 30
TIME(minutes)
FIGURE 6 Effect of aspirin on thrombin formation and secre-tion of PF4 during blood clotting. Blood was drawn from a normal donorwho had ingested 1,800 mg of aspirin during
the preceding 24 h; it was immediately placed into poly-propylene tubes. Serial samples were removed and assayed for thrombin (0) orPF4 (A).
onstrated that thrombin is the primary stimulus for platelet secretion during blood clotting. We have pre-viously shown that platelet secretion of thealpha gran-ule protein, PF4, closely parallels thrombin formation during in vitro clotting of normal blood (3). We have extended these observations in studies of the platelet release reaction in which blood from two patients with FactorV deficiency was used. Inaddition to delayed anddecreased thrombinformation,adelayinthe plate-let release reaction wasobserved inthe bloodof each patient. Inboth instances, no secretion of PF4 occurred above that seen in theanticoagulatedcontrol samples before thrombin was formed. Inasmuch asFactorVis required only for the last step in coagulation, pro-thrombin activation, the delay in the platelet release reaction mustbecaused by impaired thrombin forma-tion rather than by another coagulation factor whose activation precedes that ofprothrombin. That PF4 se-cretionbecomes normal when the abnormalitiesin pro-thrombin activation are corrected is evidence for a direct relationship between thrombin formation and platelet secretion.
Notonly were there differences in theresults ofthe patients' studies compared to normal subjects, but in addition, the results from the two patients differed markedly fromeach other. Patient E.N. had a consider-ably longer delay in both prothrombin activation and platelet secretion than patient K.H., although each had
0%plasmaFactorVand similarabnormalities intheir conventional coagulation laboratorytests. Patient E.N. also had a more striking clinical history than Patient K.H.;she hadrequired transfusion therapy for bleeding
necessitated by obstetrically related hemorrhage (9). The explanation for the differences in thrombin for-mation between the two patients is unknown, as is the relationship ofthese observations to the differences in theclinical severity of their disease. Miletich et al. (9) studied the binding of ['251]Factor Xa to washed platelets from patients with Factor V deficiency, in-cluding thetwo patients we have studied, and observed decreased binding in each case. A more marked ab-normality was observedwith Patient E.N.'s platelets, however. She had 0% of normal binding of [1251]Factor Xa to her platelets, whereas Patient K.H. had 15% of normal binding. These authors suggested that the dif-ferences in Factor Xa bindingwere a result of differ-ences in the severity of their platelet Factor V de-ficiency.Patient E.N.hadnoplateletFactorVactivity. The differences we observed in the whole blood of thesetwo patients in contrast tothe absence ofmarked differences in their plasma coagulation abnormalities isconsistent with this hypothesis. It will be necessary for us to study additional patients with Factor V de-ficiency before the relevance of these laboratory ab-normalities tothe pathophysiology ofthis disease can bedetermined.
Additional evidence fortheessential roleofthrombin in the platelet releasereaction induced by blood clot-ting wasobtained whenthrombin activity wasinhibited directlyby eitherhirudinoranti-thrombin Fab.Ineach experiment, a notable delay occurred in the clotting of fibrinogen and the release of PF4 from platelets. Althoughwe were unableto measure theendogenous thrombin concentrations in these experiments, the specificity of these inhibitors for thrombin indicates that inhibitionof thrombin was responsible for the ab-normal platelet release of PF4.
Theplateletsecretion that
eventually
occurredprob-ably did so because the concentrations of hirudin or
anti-thrombin Fabwereinsufficienttoinhibitthehigh concentrations of thrombin formedatthis stageof pro-thrombin activation (4). Also, failure to obtain more
markedinhibition with the anti-thrombin Fab may be explained byagreater
affinity
of thrombin forplatelets
than the antibody.
Although considerable evidence exists to suggest a
physiologically important role for platelets in the
ac-tivationofthecoagulationsystem(1, 2, 16),the precise mechanismby whichthisoccurs isunknown.Previous
studies indicate thatplateletsmustbeactivated before theirprocoagulantactivity becomes available(17).The nature of the physical or chemical
changes,
orboth,
that constitute "activation"is
poorly understood,
how-ever. Factor Xa binds to washedplatelets only
after they have undergone the release reaction(16).
Onceplatelet Factor Vhas been secreted it serves to bind Factor Xa to the platelet. The relevance of these observations to
physiological
coagulation
isunclear,
since Factor V is normally present in plasma. More-over, evidence has been provided that activation of platelets with ADP or collagen does not shorten the clottingtimeof platelet-rich plasma comparedtoplasma containing aphospholipid substitute for platelets (18). There are several possible explanations for our ob-servation that prothrombin activation occurs normally in spite of marked inhibition of the platelet release reaction. It may be that rather than the release reac-tion, aconformational changeinthe platelet membrane is sufficient for activation. We have no information as yet on the effect of platelet inhibitors on changes in either the platelet membrane or shape in our experi-ments.Alternatively, the platelet release reaction may be required for procoagulant activity, but a small amount of release may be sufficient to provide this activity. Another possibility is that procoagulant sub-stances are released by a mechanism different from thatwhich releases alphagranules,and that release of procoagulant substances was not blocked in our experi-ments.Therelease of Factors V and VIII from platelets appears tobe similarto that of alphagranule contents (16, 19, 20). However, the significance of this observa-tion relative to platelet procoagulant activity is unknown.
The differencesinwhole blood clottingbetween the twopatients with Factor V deficiency suggest that the smallamountofFactorVactivitycontained inPatient K.H.'s platelets (9) may have partially corrected the abnormalities in thrombingeneration associated with her plasma Factor V deficiency. The importance of this observationinrelationtonormal blood clotting, when normal amounts of plasma Factor V are present, is unknown, however. Moreover,thequestion remains as to whatinitiates plateletactivation, if this is required beforeplateletsare abletoprovide their procoagulant activity. Determination of the role of platelets in the coagulation scheme awaits characterization of the na-ture ofplatelet procoagulant activityand a demonstra-tionof how this activity becomes available under phys-iological circumstances.
ACKNOWLEDGMENTS
The authors wish to thank Ms. Linda Krentz for her expert technical assistance. Also, we would like to thank Doctors Sandra Schiffman, Donald Feinstein, Philip Majerus, and Douglas Triplett forassistingus in thestudy ofthe patients with FactorV deficiency and for the use of their laboratory
facilities.
Thisworkwassupported bygrantHL 21403(Dr. Shuman) and the Medical Research Service, Veterans Administration (Dr. Levine).
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thrombin, collagenor A23187. Blood. 50: 915-926. 11. Kaplan,K.L.,M.J.Broekman,A.Chernoff,G. R.Lesznik,
and M. Drillings. 1979. Plateletalpha-granule proteins:
studies on release and subcellular localization. Blood. 53: 604-618.
12. Charo, I. F., R. D. Feinman, andT. C. Detwiler. 1977. Interrelations of platelet aggregation and secretion. J. Clin. Invest. 60: 866-873.
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