EMILIN2 Regulates Platelet Activation,Thrombus Formation,and Clot Retraction

Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. In vitro clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.     

In vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid

We discovered recently in vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid. The cytotoxicity assays showed that ferulic acid (～300 μg/mL) did not cause any significant toxicity on three cell lines, platelets, leukocytes, and erythrocytes. In vitro assays showed inhibitory effects of ferulic acid on thrombin (THR)‐ or collagen/epinephrine‐stimulated platelet activation by inhibiting platelet aggregation, and decreasing clot retraction activity. The in vitro effect of ferulic acid on THR‐stimulated platelet activation was proved by the decrease in the secretion of serotonin from the platelets. The anticoagulant effects of ferulic acid were confirmed by the prolongation of the intrinsic or/and extrinsic pathways and the delay of recalcification time in plasma coagulation. Ferulic acid had antithrombotic effect in acute thromboembolism model in vivo, and decreased the expression of α_IIbβ₃/FIB and phosphorylation of AKT in THR‐stimulated platelet activation in vivo, and their antithrombotic efficacies hold promise for therapeutic targeting in our ongoing studies.     

Effects of forskolin and cilostazol on clot retraction

We examined the effects of two inhibitors of aggregation of human platelets the, Forskolin and Cilostazol on clot retraction. Both substances suppressed clot retraction in a dose-dependent way. Both suppress platelet aggregation because of an increase in intercellular cyclic AMP, but there was no close correlations were shown between suppression rate for clot retraction and cyclic-AMP content in platelets in the clot in each substance. Furthermore, although it has been considered that actomyosin in platelets is a major contractile source for clot retraction and that failure of actin polymerization results suppression of clot retraction. As it was difficult to obtain active actin from platelets of the reagents on the polymerization. Cilostazol accelerated actin polymerization, whereas Forskolin did not. From these findings, it was considered that the effects of both substances on clot retraction could not be interpreted directly just by the increasing effect for intracellular cyclic-AMP. Clot retraction is consider to be a in vitro model of hemostasis and its contractile force is supplied from platelets (1,2,3). Experiments used prostaglandin E-1 revealed that elevation of cyclic-AMP (c-AMP) would regulate the clot retraction, and experiments used cytochalasin B demonstrated that actomyosin is responsible to the retraction (4,5). Many date demonstrate that elevation of c-AMP level suppresses platelet aggregation (6,7). c-AMP, therefore, should play a key role on platelet activation. Forskolin and Cilostazol are newly-developed reagents as a suppress for platelet functions. Pharmacological action of these substances have been interpreted to process increase effect for intracellular c-AMP of platelets(8,9). If so, both substances should show some effect on clot retraction. Under this assumption, we examined the effects.     

Development of platelet contractile force as a research and clinical measure of platelet function

This article reviews work performed at the Medical College of Virginia of Virginia Commonwealth University during the development of a whole-blood assay of platelet function. The new assay is capable of assessing platelet function during clotting and thus allows measurement of the contribution of platelets to thrombin generation. Because platelets are monitored in the presence of thrombin, the test gages platelets under conditions of maximal activation. Three parameters are simultaneously assessed on one 700-μL sample of citrated whole blood. Platelet contractile force (PCF), the force produced by platelets during clot retraction, is directly measured as a function of time. This parameter is sensitive to platelet number, platelet metabolic status, glycoprotein IIb/IIIa status, and the presence of antithrombin activities. Clot elastic modulus (CEM), also measured as a function of time, is sensitive to fibrinogen concentration, platelet concentration, the rate of thrombin generation, the flexibility of red cells, and the production of force by platelets. The third parameter, the thrombin generation time (TGT) is determined from the PCF kinetics curve. Because PCF is absolutely thrombin dependent (no thrombin—no force), the initial upswing in PCF occurs at the moment of thrombin production. TGT is sensitive to clotting factor deficiencies, clotting factor inhibitors, and the presence of antithrombins, all of which prolong the TGT and are known to be hemophilic states. Treatment of hemophilic states with hemostatic agents shortens, the TGT toward normal. TGT has been demonstrated to be shorter and PCF to be increased in coronary artery disease, diabetes mellitus, and several other thrombophilic states. Treatment of thrombophilic states with a variety of heparin and nonheparin anticoagulants prolongs the TGT toward normal. The combination of PCF, CEM, and TGT measured on the same sample may allow rapid assessment of global hemostasis and the response to a variety of procoagulant and anticoagulant medications.     

Peroxynitrite may affect fibrinolysis via the reduction of platelet-related fibrinolysis resistance and alteration of clot structure

We tested the hypothesis that in vitro peroxynitrite (ONOO⁻, a product of activated inflammatory cells) may affect fibrinolysis in human blood through the reduction of platelet-related fibrinolysis resistance. It was found that ONOO⁻ (25–300 µM) accelerated lysis of platelet-fibrin clots (in PRP) dose-dependently, whereas fibrinolysis of platelet-free clots was slightly inhibited by ≥1000 µM stressor. Concentrations of ONOO⁻ affecting the lysis of platelet-rich clots, inhibited clot retraction (CR) in a dose-dependent manner. Thromboelastometry (ROTEM) measurements performed in PRP showed that treatment with ONOO⁻ (threshold conc. 100 µM) prolongs clotting time, and reduces alpha angle, and clot formation velocity parameters indicating for reduced thrombin formation rate. In PRP, ONOO⁻ (threshold conc. 100 µM) reduced the collagen-evoked exposure of phosphatidylserine (PS) on platelets’ plasma membrane, the shedding of platelet-derived microparticles (PMP), and inhibited platelet-dependent thrombin generation (measured in artificial system), dose-dependently. As judged by confocal microscopy, similar ONOO⁻ concentrations altered the architecture of clots formed in collagen-treated PRP. Clots formed in the presence of ONOO⁻ were less dense and were composed of thicker fibers, which make them more susceptible to lysis. In platelet-depleted plasma, ONOO⁻ (up to milimolar concentration) did not alter clot structure. Blockage of PS exposed on platelets resulted in an alteration of clot architecture toward more prone to lysis. ONOO⁻, at lysis-affecting concentrations, inhibited the collagen-evoked secretion of fibrinolytic inhibitors from platelets. We conclude that physiologically relevant ONOO⁻ concentrations may accelerate the lysis of platelet-fibrin clots predominantly via downregulation of platelet-related mechanisms including: platelet secretion, clot retraction, platelet procoagulant response, and the alteration in clot architecture associated with it.     

Similarities between platelet contraction and cellular motility during mitosis: role of platelet microtubules in clot retraction.

The effects of inhibitors of mitosis, energy metabolism and protein synthesis on clot retraction were investigated. The results show that (1) Incubation of colchicine (0-01-0-1 mM) with platelet-rich plasma (PRP) inhibits the subsequent retraction of clots derived from diluted PRP. (2) Inhibition of clot retraction by high concentrations of colchicine (up to 40 mM) can be overcome by increasing the platelet concentration in the system. (3) Incubation of clots in colchicine or 80% D2O solutions inhibits their retraction. Exposure of partially retracted clots to these agents is without effect. (4) Hydrostatic pressure retards clot retraction. (5) Incubation of PRP with either 2-deoxy-D-glucose or antimycin alone does not affect clot retraction, but a combination of these agents is inhibitory. (6) Clot retraction is not inhibited by puromycin or cycloheximide. (7) Platelets in retracting clots have constricted regions containing microfilaments and pseudopods containing microtubules. Fibrin strands are progressively condensed around the constricted regions as retraction advances. (8) The development of platelet constriction, platelet pseudopods and the intracellular microfilaments are delayed in colchicinized clots, corresponding to the retardation of retraction. Following the initial delay of retraction colchicinized clots, like controls, show condensation of fibrin strands adjacent to these constricted areas of platelets containing microfilaments. The formation of pseudopods is impaired and no microtubules are found in platelets in the presence of colchicine. The above results suggest that the thrombin-induced platelet contraction during clot retraction is a coordinated movement, which, under optimal conditions involves both microtubules and microfilaments. The contraction of microfilaments produces the constriction of platelets and brings about clot retraction by reducing the angle between fibrin strands. Platelet microtubules are related to the development of pseudopods and play a supplementary role in facilitating microfilament-mediated cellular constriction. The similarities between platelet contraction and cellular motility in mitosis is discussed.     

Separable function of platelet release reaction and clot retraction

Amiloride, a known Na+/H+ exchange inhibitor, inhibited platelet serotonin release in a dose-dependent manner (100 microM for 50% inhibition, and 1mM for the nearly complete inhibition), although amiloride (1mM) accelerated clot retraction when it was measured at decreased platelet concentration. On the contrary, cytochalasin B (10 micrograms/ml) accelerated platelet serotonin release, but it inhibited clot retraction. These results demonstrate that release reaction and clot retraction, both of which are important processes involved in platelet activation, can be functionally separated.     

Serine/threonine phosphatases regulate platelet αIIbβ3 integrin receptor outside-in signaling mechanisms and clot retraction

Aims

We studied the role of serine/threonine phosphatases (PSTPs) on α_IIbβ₃ signaling and the potential selectivity of the level of PSTP inhibition with okadaic acid (OA) on α_IIbβ₃ signaling for regulation of platelet aggregation and clot retraction.

Main methods

We used washed platelets from normal donors and OA as inhibitor of PSTPs. Clot retraction was induced by 1 U/mL of thrombin. Reorganized cytoskeleton was isolated from Triton X-100 lysed platelets. The presence of proteins incorporated to the cytoskeleton was assayed by immunoblotting with specific antibodies.

Key findings

We found that both 100 and 500 nM OA blocked platelet mediated clot retraction. In contrast, only 500 nM OA inhibited thrombin-induced inside-out α_IIbβ₃ activation, platelet aggregation, and cytoskeletal reorganization. Among markers of α_IIbβ₃ outside-in signaling, 500 nM OA inhibited the incorporation to the cytoskeleton of syk, src, and FAK (Focal Adhesion Kinase) tyrosine kinases and the incorporation and phosphorylation at Tyr⁷⁵⁹ of the β₃ chain of α_IIbβ₃, while 100 nM OA only inhibited the FAK translocation and its tyrosine phosphorylation.

Significance

The level of inhibition of PSTPs by low or high OA concentration (33% and 73% inhibition, respectively) in intact whole cells differentially regulates platelet aggregation and integrin signaling, but have a common effect in blocking clot retraction. The latter may be associated with the presence of phosphorylated FAK in the cytoskeleton. This study reveals a novel target for anti-platelet treatment to block clot retraction without affecting the platelet hemostatic function by a partial inhibition of PSTPs.     

Clotting of citrated plasma by a proteolytic enzyme associated with an antibacterial agent from serum

Two to 4 mg/ml of an antibacterial agent occurring in the serum of humans and animals caused the clotting of citrated rabbit, human, calf, ox, sheep, goat, guinea pig, rat, mouse, horse, chicken, pigeon and swine plasma. Heparinized plasmas of the same species were found resistant to the clotting action of the antibacterial agent. Citrated plasma previously heated at 56 C for 30 min, treated with 640 units/ml tyrosinase for 60 min, or absorbed with 0.2M Ca₃(PO₄)₂ and 0.2M Mg(OH)₂ was also found resistant to the clotting action of the antibacterial agent. The clotting action of the antibacterial agent was not affected by heating at 66 C for 60 min, nor by multiple passage through Seitz filters. The coagulation of citrated plasma proceeded most rapidly with 0.2M Tris(hydroxymethyl)-amino-methane buffer, pH 7 to 9, or distilled water as the antibacterial agent solvent; 0.2M phosphate buffer, pH 6 to 8, reduced the clotting action of the antibacterial agent, while 0.2M citrate-phosphate buffer, pH 4.0, or 0.2M carbonate-bicarbonate buffer, pH 10.0, inhibited entirely the clotting activity of this agent.     

Mechanism of Coagulation in Gecarcinus lateralis

Agglutination of cells, degranulation, and loss of cellularmembranes compose the major form of coagulation in the hemolymphof Gecarcinus lateralis. It is only after agglutination of theformed elements of the hemolymph that fibrin-like strands appear.Sodium citrate, in a concentration of 10% or more to preventcoagulation, is always inadequate to prevent cell agglutination. Multiple studies by protein electrophoresis failed to revealany differences between plasma and serum, nor did they allowus to identify a soluble protein in plasma that did not appearin serum. Crab hemolymph changes in its capacity for clottingduring the molt cycle, with the most rapid clotting occurringin the premolt period. A new protein appears in the premoltperiod, but its relation to the whole clotting mechanism isunknown. In contradistinction to vertebrate systems, citrated hemolymphdoes not clot when calcium is added. There is no relationshipthat can be demonstrated between activating systems in vertebrateplasma and clotting in the crab. It would seem that, ratherthan the vertebrate coagulating system evolving from the crustaceantype of clotting system, the development of these clotting systemshas run in parallel. The crustacean cell, in addition, appearsto be more potent than vertebrate cells in clotting systems.The comparison of human lymph to crustacean hemolymph wouldindicate that, for a given amount of cells, crustacean hemolymphclots 2 to 20 times faster than human lymph. On the other hand,agglutination of cells is a fundamental initiating step in coagulationof both human blood and crustacean hemolymph.     

Thrombosis Prevention by Acetylsalicylic Acid in Hyperlipemic Rats

In rats, administration of acetylsalicylic acid (ASA) by stomach tube two hours before blood removal, or addition of the drug to platelet-rich plasma in vitro, markedly inhibited platelet aggregation induced by thrombin, ADP and collagen. Addition of ASA in vitro to human platelet-rich plasma also inhibited platelet aggregation by thrombin, ADP and collagen. In hyperlipemic rats, ASA (100 to 200 mg./kg.), administered by stomach tube once or five times, markedly inhibited the production of thrombosis initiated by intravenous injection of S. typhosa endotoxin. In these experiments, thrombosis prevention by ASA was associated with both a decrease in platelet aggregation and an increase in the recalcification plasma clotting time.     

Identification of a novel binding site for platelet integrins alpha IIb beta 3 (GPIIbIIIa) and alpha 5 beta 1 in the gamma C-domain of fibrinogen

The interactions of platelets with fibrinogen mediate a variety of responses including adhesion, platelet aggregation, and fibrin clot retraction. Whereas it was assumed that interactions of the platelet integrin alpha IIb beta 3 with the AGDV sequence in the gamma C-domain of fibrinogen and/or RGD sites in the A alpha chains are involved in clot retraction and adhesion, recent data demonstrated that fibrinogen lacking these sites still supported clot retraction. These findings suggested that an unknown site in fibrinogen and/or other integrins participate in clot retraction. Here we have identified a sequence within gamma C that mediates binding of fibrinogen to platelets. Synthetic peptide duplicating the 365-383 sequence in gamma C, designated P3, efficiently inhibited clot retraction in a dose-dependent manner. Furthermore, P3 supported platelet adhesion and was an effective inhibitor of platelet adhesion to fibrinogen fragments. Analysis of overlapping peptides spanning P3 and mutant recombinant gamma C-domains demonstrated that the P3 activity is contained primarily within gamma 370-383. Integrins alpha IIb beta 3 and alpha 5 beta 1 were implicated in recognition of P3, since platelet adhesion to the peptide was blocked by function-blocking monoclonal antibodies against these receptors. Direct evidence that alpha IIb beta 3 and alpha 5 beta 1 bind P3 was obtained by selective capture of these integrins from platelet lysates using a P3 affinity matrix. Thus, these data suggest that the P3 sequence in the gamma C-domain of fibrinogen defines a previously unknown recognition specificity of alpha IIb beta 3 and alpha 5 beta 1 and may function as a binding site for these integrins.     

Impaired platelet aggregation and sustained bleeding in mice lacking the fibrinogen motif bound by integrin alpha IIb beta 3.

Blood loss at sites of vascular rupture is controlled by the adhesion and aggregation of platelets and the formation of an insoluble fibrin matrix. Fibrinogen is considered to be critical in these processes by both providing an abundant dimeric ligand for alpha IIb beta 3-mediated platelet aggregation, and serving as the fundamental building block of the fibrin polymer. To establish an in vivo model system to examine in detail the importance of alpha IIb beta 3-fibrinogen interactions in platelet function, hemostasis, response to injury and vasoocclusive disease, and to test the prevailing hypothesis that the C-terminal segment of the fibrinogen gamma chain is essential for alpha IIb beta 3 binding, we have used gene-targeting technology in mice to eliminate the last five residues (QAGDV) from the gamma chain. Mice homozygous for the modified gamma chain gene (gamma delta 5/gamma delta 5) displayed a generally normal hematological profile, including normal platelet count, plasma fibrinogen level, clotting time and fibrin crosslinking. However, both gamma delta 5-fibrinogen binding to alpha IIb beta 3 and platelet aggregation were highly defective. Remarkably, another alpha IIb beta 3-dependent process, clot retraction, was unaffected by the gamma delta 5 mutation. Despite the preservation of clotting function, gamma delta 5/gamma delta 5 mice were unable to control blood loss following a surgical challenge and occasionally developed fatal neonatal bleeding events.     

Integration of Acoustic Radiation Force and Optical Imaging for Blood Plasma Clot Stiffness Measurement

Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood’s transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 μm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties.     

Reorganization of contractile elements in the platelet during clot retraction

Electron microscopic studies have been carried out on human platelets in the clot retraction. In the early stage of clot formation, platelets send out filopodia, in which thin filaments run longitudinally. The thin filaments are often observed to attach to the cell membrane where fibrin strands bind from the extracellular surface. In the later stage of clot formation, thick filaments become observable, mainly in the cell body of the platelets. These thick filaments are arranged to form an ordered array, and thin filaments run parallel to them. The thin filaments often attach to the end of the thick filaments. However, thin filaments are not seen between the arrays of thick filaments. Similar structures are also observed in the cytoskeleton of the contracted platelet. These filaments closely resemble the purified myosin aggregates formed under low ionic strength. Thus, during clot retraction, both actin and myosin in platelets are reorganized into thin and thick filaments, respectively.     

Studies on platelet functions in hirudin plasma

Comparative studies on platelet responses in citrated, hirudin and heparin plasma were carried out. The adhesion of 111In-labelled rabbit platelets to the subendothelium of rabbit aorta was more pronounced in hirudin plasma than in heparin and citrated plasma. There were no significant differences in the collagen-induced aggregation and secretion of 14C-serotonin of human blood platelets in the three plasma samples. The extent of the ADP-induced aggregation was nearly the same in the three plasma samples, however, the aggregation was reversible in hirudin plasma. Adrenaline induced a small primary aggregation in hirudin plasma whereas in citrated and in heparin plasma the aggregation was a biphasic one. Secretion of 14C-serotonin induced by ADP or adrenaline occurred in citrated plasma only. Hirudin proved to be a suitable anticoagulant for studying platelet functions at physiological calcium concentrations.     

Biophysical mechanisms of contact activation of blood-plasma clotting

This review considers the biochemical and biophysical mechanisms that trigger blood clotting upon contact of blood with an alien surface and leads via a cascade of enzymatic reactions to fibrin polymerization and the formation of a blood plasma clot, which permeates a primary platelet aggregate to produce a dense hemostatic clot. In spite of the substantial number of experimental and theoretical studies on the subject, there is still no consistent opinion as to what processes occur as the blood plasma contacts a surface. This review discusses the role that plasma protein factor XII and various surfaces play in triggering the contact pathway in vivo and in vitro. Current views of the molecular events that underlie the process are described.     

Effect of RA233 on Platelet Function In Vitro

RA233, a new pyrimido-pyrimidine compound, is a powerful inhibitor of platelet function tested in vitro; it inhibits calcium and adenosine diphosphate (A.D.P.)-induced platelet aggregation, inhibits the retention of platelets by glass beads, decreases the release of platelet factor 3 by kaolin, and inhibits clot retraction. In some in-vitro systems RA233 is significantly more potent that its analogue RA433 in inhibiting platelet function.     

Thrombocytin, a serine protease from Bothrops atrox venom. 2. Interaction with platelets and plasma-clotting factors.

Thrombocytin, a serine protease from Bothrops atrox venom, caused platelet aggregation and release of platelet constituents at a concentration of 10(-7) M and clot retraction at a concentration of 2 x 10(-9) M. Thrombocytin was slightly more active when tested on platelets in plasma than on washed platelets suspended in Tyrode--albumin solution. Thrombin was 5 times more active than thrombocytin when tested on platelets in plasma and 50 times more active when tested on washed platelets. The patterns or release induced by thrombocytin and thrombin were similar. Prostaglandin E1 (10(-5) M) produced complete inhibition of platelet release induced by thrombocytin and thrombin. Indomethacin (10(-4) M) was without any effect. Antithrombin III, in the presence of heparin, inhibited the action of thrombocytin on platelets and on a synthetic peptide substrate (Tos-Gly-Pro-Arg-pNA.HCl). formation of an antithrombin III--thrombocytin complex was demonstrated on NaDodSO4--polyacrylamide gel electrophoresis. Hirudin and alpha 1-antitrypsin did not inactivate thrombocytin. Thrombocytin had a low fibrinogen-clotting activity (less than 0.06% that of thrombin). Thrombocytin also caused progressive degradation of the alpha chain of human fibrinogen, and it cleaved prothrombin, releasing products similar to intermediate 1 and fragment 1 produced by thrombin. Thrombocytin activated factor XIII by limited proteolysis and increased the procoagulant activity of factor VIII in a manner analogous to that of thrombin.     

The determination of platelet factor 3 using hirudin]

The influence of blood platelets on the recalcification time under hirudin was investigated. Contrary to the investigations of whole-blood which reveal a pathological prolongation of the hirudin tolerance test only at platelet numbers under 30,000/mug, a change of the recalcification time under hirudin could also be found in the plasma at higher platelet numbers. The recalcification time increased inversely proportionally with falling platelet number. The shortening of time in platelets rich plasma attributed to the activity of platelet factor 3. Differences in the examinations of whole-blood may be attributed to an erythrocyte activity similar to factor 3. The application of hirudin for determining platelet factor 3 is recommended as a sensible method easily to be performed in practice.