Effects of high-molecular-weight cryoprotectants on platelets and the coagulation system

The objective of this study is to examine the effects of the most widely used high-molecular-weight cryoprotectants on the coagulation system. Dextran, hydryoxyethyl starch (HES), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), and albumin were added at different concentrations in the range between 0.01-1% (w/v) to solvent/detergent-treated plasma. Using a STA/STA Compact coagulation analyzer the following clotting tests were performed: prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Factor V, and Factor VIII percentage of activity. PVP and PEG caused a significant increase in APTT, a decrease in Factor VIII percentage of activity, and a slight decrease in TT, while PT and Factor V percentage of activity remained unchanged. Dextran, HES, and albumin did not effect the clotting tests. The effect of high-molecular-weight cryoprotectants on platelets was assessed by platelet-induced clot retraction (PICR) and aggregation with thrombin and agglutination with ristocetin. Platelet aggregation and agglutination were unaffected by all cryoprotectants tested; however, PICR was significantly reduced in the presence of PVP or PEG. Possible mechanisms by which PVP and PEG interfere with the coagulation system are discussed. We also raise issues concerning the development of one-step blood cryopreservation techniques which do not require cryoprotectant removal prior to transfusion.     

The effect of cyanate on the clotting proteins and platelet function.

Cyanate reacts with unchanged amino groups of various proteins in a specific irreversible carbamylation reaction. The effect of this molecule on the clotting process and the effects of carbamylation on the clotting proteins and platelet functions were investigated in vitro. An immediate effect on the clotting proteins, not related to pH, was seen in the screening tests prothrombin time, partial thromboplastin time and thrombin time at the highest concentration (100 mM), to a lesser degree at the lower concentration (10 mM). These changes reflected decreases of 19 and 36% respectively in Factor V and X activity, an inhibition of 63-75% of Factors VII, IX, X and XI activity, and 80% inhibition of thrombin activity. The inhibitory changes of carbamylation increased with time. No changes were seen in the activity of Factors I and VIII. Platelet function studies revealed no inhibition of Factor III release; aggregation was abnormal only at high concentrations with epinephrine and collagen induction and partially reversible by resuspension in normal plasma.     

The effects of nonpenetrating cryoprotectants added to TEST-yolk-glycerol extender on the post-thaw motility of ram spermatozoa

The effects of adding five different concentrations of 17 polymeric compounds to TEST-yolk-glycerol extender on ram spermatozoa survival was studied. These were Aquacide (I, II, and III); dextran (0.8-1.6, 1.9, 15-20, 70, and 200-300 kDa); three types of Dri-Sweet; hydroxyethyl starch; methylcellulose, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, and Supercol 912. All the compounds tested except the Dri-Sweet compounds and hydroxyethyl starch significantly (P less than 0.05) decreased percentages of motile cells in unfrozen samples. The use of dextran (0.8-1.6 kDa; hydrolyzed dextran separated by ethanol) and Aquacide II significantly (P less than 0.05) increased post-thaw motility of spermatozoa frozen in pellets. Dextran (15-20 kDa), dextran (0.8-1.6 kDa), Aquacide II, and hydroxyethyl starch significantly (P less than 0.05) increased the percentages of post-thaw motility of ram spermatozoa frozen in the presence of glycerol and egg yolk.     

The effect of plasma von Willebrand factor on the binding of human factor VIII to thrombin-activated human platelets

The binding of 35S-labeled recombinant human Factor VIII to activated human platelets was studied in the presence and absence of exogenous plasma von Willebrand factor. In the absence of added von Willebrand Factor, platelets bound 210 molecules of Factor VIII/platelet when the unbound Factor VIII concentration was 2.0 nM (Kd = 2.9 nM). As the von Willebrand factor concentration was increased, the number of Factor VIII molecules bound/platelet decreased to 10 molecules of Factor VIII bound/platelet at 24 micrograms/ml of added vWF. Addition of an anti-vWF monoclonal antibody that inhibits the vWF-Factor VIII interaction attenuated the ability of vWF to inhibit binding of Factor VIII to platelets. In contrast, addition of a control anti-vWF antibody that does not block the vWF-Factor VIII interaction did not affect the ability of vWF to inhibit Factor VIII binding to platelets. From the vWF concentration dependence of inhibition of Factor VIII-platelet binding, a dissociation constant for the Factor VIII-vWF interaction was calculated (Kd = 0.44 nM). To further elucidate the role that vWF may play in preventing the interaction of Factor VIII with platelets, the platelet binding properties of a Factor VIII deletion mutant (90-73) which lacks the primary vWF-binding site was studied. The binding of this mutant was unaffected by added exogenous vWF. These observations demonstrate that Factor VIII can interact with platelets in a manner independent of vWF but that excess vWF in plasma can effectively compete with platelets for the binding of Factor VIII. In addition, since cleavage of Factor VIII by thrombin separates a vWF-binding domain from Factor VIIIa, we propose that activation of Factor VIII by thrombin may elicit release of activated Factor VIII from vWF and thereby make it fully available for platelet binding.     

Measurement of blood coagulation Factor XIIIa formation in plasma containing glycyl-L-prolyl-L-arginyl-L-proline

A method to directly measure the formation of blood coagulation Factor XIIIa in platelet-poor plasma unmodified by heat is described. The synthetic peptide glycyl-L-prolyl-L-arginyl-L-proline, a fibrin-polymerization inhibitor, was used to prevent clotting of platelet-poor plasma. Plasma was diluted to a final concentration of 2.5% (v/v) in 0.1 M Tris-HCl, pH 8.5, buffer containing 25% glycerol, 5 mM calcium chloride, and 0.25 mM glycyl-L-prolyl-L-arginyl-L-proline and then activated by thrombin (20 U/ml) for 15 min. The Factor XIIIa-catalyzed incorporation of [3H]putrescine into Hammersten casein was used to measure Factor XIIIa formation. The assay detected Factor XIIIa in 2.5 to 50 microliter of thrombin-treated plasma. When purified Factor XIII was added to Factor XIII-deficient plasma, there was complete recovery of the Factor XIII added. Glycyl-L-prolyl-L-arginyl-L-proline did not inhibit Factor XIIIa activity in thrombin-treated plasma or purified platelet Factor XIIIa. Glycerol stabilized Factor XIIIa activity in thrombin-treated plasma and buffer for 60 min. The presence of fibrinogen in plasma did not modify the assay results. The time course of thrombin-catalyzed Factor XIIIa formation in platelet-poor plasma containing glycyl-L-prolyl-L-arginyl-L-proline was directly measured using the assay.     

Platelet-derived microparticles express high affinity receptors for factor VIII.

Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va.     

Peptide map analysis of normal plasma and platelet factor VIII antigen

Factor VIII antigen from platelet intracellular granules was immunoprecipitated using a monospecific rabbit antibody to normal plasma factor VIII antigen. The factor VIII antigen in the immunoprecipitate was isolated on sodium dodecyl sulfate polyacrylamide gels, radiolabeled with ¹²⁵I, trypsinized, and subjected to peptide mapping using two dimensional high voltage electrophoresis and thin layer chromatography. The platelet protein was compared to purified plasma factor VIII antigen. The two dimensional tryptic ¹²⁵I peptide map of platelet granule factor VIII antigen was similar but not identical to the plasma factor VIII antigen peptide map. The platelet and plasma proteins shared approximately 34 radioactive peptide spots. Seven plasma factor VIII antigen peptides were not detected in platelet factor VIII antigen. The reason for the structural differences of plasma and platelet granule factor VIII antigen are unknown. The possibility is raised that proteolysis has altered the platelet protein in vitro. It is also possible that factor VIII antigen synthesized by megakaryocytes differs from the plasma protein.     

Blood coagulation studies in normothermic,hibernating, and aroused Spermophilus franklini

The blood coagulation system of Spermophilus franklini was evaluated from normothermic, hibernating, and aroused individuals. Clotting time, thrombin time, prothrombin time, and partial thromboplastin time were measured to test the state of coagulability. The concentrations of the formed elements and the titers of five plasma factors were also determined.During hibernation, clotting time significantly increased above normothermic levels. Arousal resulted in clotting time returning toward normothermic values. Both thrombin time and partial thromboplastin time significantly increased above normothermic levels in blood from hibernators. The two tests exhibited normothermic levels in arousing individuals. Prothrombin time did not increase in blood from hibernating animals.Erythrocytes, leukocytes, and platelets were found to be significantly reduced in number in hibernating animals. Leukocyte and platelet numbers returned to normothermic levels during arousal.Factor VII, Factor X, prothrombin, and heparin concentrations did not significantly change from normothermic levels in hibernating individuals. Factor V, however, displayed a 45% decrease in concentration in hibernating individuals, with arousal resulting in near-normothermic levels. Aroused individuals displayed a doubling of prothrombin concentrations relative to normothermic individuals.     

The binding of 35S-labeled recombinant factor VIII to activated and unactivated human platelets

Recombinant-derived human Factor VIII was labeled intrinsically with [35S]methionine, and its binding to washed human platelets was studied. Binding measurements were performed by incubating Factor VIII and platelets for 15 min at room temperature in Tyrode's solution supplemented with Ca2+ (5.0 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (5.0 mM), 0.50% bovine serum albumin, and the Factor Xa and thrombin inhibitors 5-dimethylaminonaphthalene-1-sulfonylglutamylglycinylarginyl chloromethyl ketone and 5-dimethylaminonaphthalene-1-sulfonyl-arginine-N-(3-ethyl-1, 5-pentanediyl)amide. Separation of free from bound Factor VIII was accomplished by centrifugation through oil, and nonspecific binding was determined with excess unlabeled Factor VIII. Binding was saturable, reversible, and stimulated 20-fold after platelet activation with thrombin. Furthermore, binding was specific in that bound labeled Factor VIII could be displaced by excess unlabeled Factor VIII, but not by Factor V. Scatchard analysis indicated a single class of binding sites with Kd = 2.9 nM and 450 sites/activated platelet. The time course of displacement indicated a t1/2 of bound Factor VIII of approximately 5 min. When platelets were incubated in Ca2+, both the heavy and light chains of Factor VIII were bound, whereas exposure to EDTA resulted in the binding of the light chain only. These results demonstrate the specific reversible binding of Factor VIII to human platelets, likely mediated through the light chain.     

Fluorescence properties and functional roles of tryptophan residues 60d, 96, 148, 207, and 215 of thrombin

Conservative Trp-to-Phe mutations were individually created in human thrombin at positions 60d, 96, 148, 207, and 215. Fluorescence intensities for these residues varied by a factor of 6. Residues 60d, 96, 148, and 215 transferred energy to the thrombin inhibitor 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5- pentanediyl)amide efficiently, but residue 207 did not. Intensities correlated inversely with exposure to solvent, and measured and theoretical energy transfer efficiencies agreed well. Function was measured with respect to fibrinogen clotting, platelet and factor V activation, inhibition by antithrombin, and the thrombomodulin-dependent activation of protein C and thrombin-activable fibrinolysis inhibitor (TAFI). All activities of W96F and W207F ranged from 74 to 154% of the wild-type activity. This was also true for W148F, except for inhibition by antithrombin, where it showed 60% activity. W60dF was deficient by 30, 57, and 43% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1006)), respectively. W215F was deficient by 90, 55, and 56% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1536)). With protein C and TAFI, W96F, W148F, and W207F were normal. W60dF, however, was 76 and 23% of normal levels with protein C and TAFI, respectively. In contrast, W215F was 25 and 124% of normal levels in these reactions. Thus, many activities of thrombin are retained upon substitution of Trp with Phe at positions 96, 148, and 207. Trp(60d), however, appears to be very important for TAFI activation, and Trp(215) appears to very important for clotting and protein C activation.     

Combined deficiency of factor V and factor VIII. A report of another case.

A patient with combined factor V and factor VIII deficiency is presented. The bleeding manifestations were: easy bruising, post-traumatic bleeding, bleeding after tooth extractions. The main laboratory feature was a prolonged partial thromboplastin time which was corrected by the addition of adsorbed normal plasma but not by the addition of normal serum, hemophilia A plasma of another patient with combined factor V and factor VIII deficiency. The thromboplastin generation test was clearly abnormal and was corrected by the addition of adsorbed normal plasma but not by addition of normal serum. Prothrombin consumption was also defective. Prothrombin time was slightly prolonged too, Thrombin time, platelet and vascular tests were within normal limits and there was no hyperfibrinolysis. Factor VIII was 8% of normal, whereas factor V was 14% of normal. Factor VIII associated antigen was normal. All other clotting factors were within normal limits. The parents of the propositus were consanguineous (first cousins) but had normal factor V and factor VIII activity and normal factor VIII antigen. The same was true for other family members. The hereditary transmission of the condition appears autosomal recessive.     

Characterization of the fibrin polymer structure that accelerates thrombin cleavage of plasma factor XIII

The effect of plasmin-derived fibrin(ogen) degradation products on alpha-thrombin cleavage of plasma Factor XIII was studied to identify the fibrin polymer structure that promotes Factor XIIIa formation. Fibrin polymers derived from fibrinogen and Fragment X enhanced the rate of thrombin cleavage of plasma Factor XIII in plasma or buffered solutions. The concentrations of fibrinogen and Fragment X that promoted half-maximal rates of Factor XIIIa formation were 5 and 40 micrograms/ml, respectively. Fragments Y, D, E, D-dimer, and photooxidized fibrinogen did not enhance thrombin cleavage of Factor XIII. Although purified Fragment D1 inhibited fibrin gelation, the soluble protofibrils promoted thrombin activation of Factor XIII. Noncrosslinked fibrin fibers failed to enhance thrombin cleavage of Factor XIII. In conclusion, soluble fibrin oligomers function to promote thrombin cleavage of plasma Factor XIII during blood clotting.     

Enhancement of Pig Embryonic Implants in Factor VIII KO Mice: A Novel Role for the Coagulation Cascade in Organ Size Control

Very little is known about the mechanisms that contribute to organ size differences between species. In the present study, we used a mouse model of embryonic pig tissue implantation to define the role of host Factor VIII in controlling the final size attained by the implant. We show here that pig embryonic spleen, pancreas, and liver all grow to an increased size in mice that are deficient in the Factor VIII clotting cascade. Similar results were obtained using the transplantation model after treatment with the low molecular weight heparin derivative Clexane which markedly enhanced transplant size. Likewise, enhanced size was found upon treatment with the direct thrombin inhibitor Dabigatran, suggesting that organ size regulation might be mediated by thrombin, downstream of Factor VIII. Considering that thrombin was shown to mediate various functions unrelated to blood clotting, either directly by cleavage of protease-activated receptors (PARs) or indirectly by cleaving osteopontin (OPN) on stroma cells, the role of PAR1 and PAR4 antagonists as well as treatment with cleaved form of OPN (tcOPN) were tested. While the former was not found to have an impact on overgrowth of embryonic pig spleen implants, marked reduction of size was noted upon treatment with the (tcOPN). Collectively, our surprising set of observations suggests that factors of the coagulation cascade have a novel role in organ size control.     

Platelet - vessel wall interaction: role of blood clotting

Vascular damage initiates not only the adhesion and aggregation of blood platelets but also coagulation, which is of mixed (intrinsic and extrinsic) origin. Evidence is presented that thrombin, generated as a result of the injury, is a prerequisite for platelet aggregation. Platelets, after activation, in their turn promote coagulation. Prostaglandin I2 (PGI2 or prostacyclin) inhibits coagulation induced by damaged vascular tissue. This effect of PGI2 is mediated by the inhibition of platelets in their participation in the generation of factor Xa and thrombin. Dietary cod liver oil, by changing plasma coagulability, decreases the procoagulation activity of vessel walls, and arterial thrombosis. Another fish oil with similar effects on plasma coagulability and some other haemostatic parameters does not modify vessel wall-induced clotting, nor does it significantly lower arterial thrombosis tendency; this indicates the physiological relevance of vessel wall-induced clotting in arterial thrombus formation. Some evidence is also given for the importance of vessel wall-induced clotting in primary haemostasis.     

A thrombin-based peptide corresponding to the sequence of the thrombomodulin-binding site blocks the procoagulant activities of thrombin.

Thrombomodulin, a cofactor in the thrombin-catalyzed activation of protein C, blocks the procoagulant activities of thrombin such as fibrinogen clotting, Factor V activation, and platelet activation. The binding site for thrombomodulin within human thrombin has been localized at a region comprising residues Thr147-Ser158 of the B-chain of thrombin. The dodecapeptide sequence, TWTANVGKGQPS, corresponding to these residues inhibits thrombin binding to thrombomodulin with an apparent Ki = 94 microM (Suzuki, K., Nishioka, J., and Hayashi, T. (1990) J. Biol. Chem. 265, 13263-13267). We have found that the inhibitory effect of the dodecapeptide on the thrombin-thrombomodulin interaction is sequence-specific, and that residues Asn151, Lys154, and Gln156 are essential for thrombomodulin binding. The dodecapeptide was also found to directly block thrombin procoagulant activities, fibrinogen clotting (concentration for half-maximum inhibition, 385 microM). Factor V activation (concentration for half-maximum inhibition, 33 microM), and platelet activation (concentration for half-maximum inhibition, 645 microM). This peptide did not block thrombin inhibition by antithrombin III, but blocked thrombin inhibition by hirudin. These findings suggest that the binding site for thrombomodulin in thrombin is shared with the sites for fibrinogen, Factor V, platelets, and hirudin, and that, therefore, the inhibition of thrombin procoagulant activities by thrombomodulin in part results from blocking of the interaction between thrombin and the procoagulant protein substrates by thrombomodulin.     

Specificity of phosphatidylserine-containing membrane binding sites for factor VIII. Studies with model membranes supported by glass microspheres (lipospheres).

Factor VIII functions in an enzyme complex upon the activated platelet membrane where phosphatidylserine exposure correlates with expression of receptors for factor VIII. To evaluate the specificity of phosphatidylserine-containing membrane binding sites for factor VIII, we have developed a novel membrane model in which phospholipid bilayers are supported by glass microspheres (lipospheres). The binding of fluorescein-labeled factor VIII to lipospheres with membranes of 15% phosphatidylserine was equivalent to binding to phospholipid vesicles (KD = 4.8 nM). Purified von Willebrand factor (vWf), a carrier protein for factor VIII, decreased membrane binding of factor VIII with a Ki of 10 micrograms/ml. Likewise, normal plasma decreased bound factor VIII by more than 90% whereas plasma lacking vWf decreased the binding of factor VIII by only 20%. Proteolytic activation of factor VIII by thrombin, which releases factor VIII from vWf, increased liposphere binding in the presence of vWf and in the presence of normal plasma. Although factor V is homologous to factor VIII and binds to lipospheres with the same affinity, purified factor V was not an efficient competitor for the membrane binding sites of factor VIII. These results indicate that phosphatidylserine-containing membrane sites have sufficient specificity to select thrombin-activated factor VIII from the range of phospholipid-binding proteins in plasma.     

Factor VIII/von Willebrand Factor has potent lectin activity

Highly-purified plasma and platelet Factor VIII/von Willebrand Factor had potent lectin activity when measured in a haemagglutination assay. This lectin activity was inhibited by monoclonal and heterologous antibodies to Factor VIII/von Willebrand Factor as well as by hexosamines, mannose and net-positively charged amino acids.     

The correlated actions of vitamins C,K, and Q

Three vitamins (C, K, and Q), two of which are unequivocally established and the existence of the third supported by both experimental and clinical evidence, are needed to prevent hemorrhagic states and therefore can be designated the hemostatic vitamins. The coordinated actions of these vitamins can be epitomized by a diagram. Vitamin K is responsible for the synthesis of four basic clotting factors that function in two distinct pathways for the activation of prothrombin to thrombin. Vitamin Q also functions in these pathways. In the intrinsic, it supplies by means of platelets the Q factor that with factors VIII and IX generates intrinsic (plasma) thromboplastin. In the extrinsic pathway, it is related to tissue thromboplastin which has the Q factor as a part of its structure. It appears to be a phospholipid obtained from exogenous sources. Both vitamins C and K have a potential redox mechanism in their structure which can be hypothecated to function in the synthesis and maintenance of mesenchymal tissue.     

Anticoagulant activities of a monoclonal antibody that binds to exosite II of thrombin.

A monoclonal IgG isolated from a patient with multiple myeloma has been shown to bind to exosite II of thrombin, prolong both the thrombin time and the activated partial thromboplastin time (aPTT) when added to normal plasma, and alter the kinetics of hydrolysis of synthetic peptide substrates. Although the IgG does not affect cleavage of fibrinogen by thrombin, it increases the rate of inhibition of thrombin by purified antithrombin approximately 3-fold. Experiments with plasma immunodepleted of antithrombin or heparin cofactor II confirm that prolongation of the thrombin time requires antithrombin. By contrast, prolongation of the aPTT requires neither antithrombin nor heparin cofactor II. The IgG delays clotting of plasma initiated by purified factor IXa but has much less of an effect on clotting initiated by factor Xa. In a purified system, the IgG decreases the rate of activation of factor VIII by thrombin. These studies indicate that binding of a monoclonal IgG to exosite II prolongs the thrombin time indirectly by accelerating the thrombin-antithrombin reaction and may prolong the aPTT by interfering with activation of factor VIII, thereby diminishing the catalytic activity of the factor IXa/VIIIa complex.     

Inhibition of factor VIII-associated platelet aggregation by heparin and dextran sulfate, and its mechanism.

Both ristocetin-induced aggregation in the presence of human factor VIII and bovine factor VIII-induced aggregation of washed normal human platelets were inhibited or reversed by the addition of heparin or dextran sulfate. These actions of dextran sulfate were stronger than those of heparin, and dependent on the sulfur content of dextran sulfate. In order to study the mechanism of actions of dextran sulfate and heparin, the affinity chromatographic experiment of factor VIII in human and bovine plasma, respectively, was carried out by using a dextran sulfate- and a heparin-Agarose column. Both human and bovine factor VIII have a strong affinity for dextran sulfate with high sulfur content and a weak affinity for heparin, but no affinity for dextran sulfate with low sulfur content. From these results, it is suggested that dextran sulfate or heparin binds directly the human and bovine factor VIII, which is an essential factor for the maintenance of the weak interplatelet bonds, and either inhibits or reverses the platelet aggregation.