High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s)

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.

High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s)

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.

Multimeric structure of platelet factor VIII/von Willebrand factor: the presence of larger multimers and their reassociation with thrombin- stimulated platelets

The multimeric structure of platelet factor VIII/von Willebrand factor (FVIII/vWF) in cell extracts and in collagen and thrombin releasates has been analyzed by SDS polyacrylamide gel electrophoresis followed by detection with 125I-anti-FVIII/vWF. Platelets contained larger multimers than those normally present in plasma. When secreted FVIII/vWF was analyzed, all platelets. In contrast, in thrombin releasates the larger multimers were lost in a manner dependent on divalent cations, time, and thrombin dose. This loss could not be accounted for by modification of FVIII/vWF by thrombin or platelet enzymes since no effect of thrombin on the multimeric structure of FVIII/vWF in the absence of platelets or in the presence of platelet lysates was observed. Large multimers of 125I-labeled purified FVIII/vWF underwent divalent cation-dependent association with platelets in the presence of thrombin, indicating that the loss of FVIII/vWF from thrombin releasates was due to reassociation with the platelet. These studies show a structural difference between platelet and plasma FVIII/vWF that suggests a specific role for platelet FVIII/vWF in hemostasis.

Multimeric structure of platelet factor VIII/von Willebrand factor: the presence of larger multimers and their reassociation with thrombin- stimulated platelets

The multimeric structure of platelet factor VIII/von Willebrand factor (FVIII/vWF) in cell extracts and in collagen and thrombin releasates has been analyzed by SDS polyacrylamide gel electrophoresis followed by detection with 125I-anti-FVIII/vWF. Platelets contained larger multimers than those normally present in plasma. When secreted FVIII/vWF was analyzed, all platelets. In contrast, in thrombin releasates the larger multimers were lost in a manner dependent on divalent cations, time, and thrombin dose. This loss could not be accounted for by modification of FVIII/vWF by thrombin or platelet enzymes since no effect of thrombin on the multimeric structure of FVIII/vWF in the absence of platelets or in the presence of platelet lysates was observed. Large multimers of 125I-labeled purified FVIII/vWF underwent divalent cation-dependent association with platelets in the presence of thrombin, indicating that the loss of FVIII/vWF from thrombin releasates was due to reassociation with the platelet. These studies show a structural difference between platelet and plasma FVIII/vWF that suggests a specific role for platelet FVIII/vWF in hemostasis.

The Role of Chelators in Surface Metabolism of Adenosine Nucleotides Added to Native Human Blood Platelets in Vitro

SummaryThe production of ATP and AMP by human platelets exposed in this study to a large excess of exogenous ADP has been confirmed, using nonanticoagulated platelet-rich-plasma. Production of ATP differed from that of AMP in that divalent cation requirements were not the same, and there were differences in sources yielding maximal production. ATP production proceeded in the absence of free calcium ions, although it was enhanced by their presence. AMP production was partially or completely arrested by full EDTA chelation of the same platelet mixture. Citrate anion regularly enhanced the net production of AMP (but not of ATP) when added to native platelet-rich-plasma, provided some free calcium ions were present. When native human platelets were resuspended in serum from autologous platelet-poor-plasma, ATP production was markedly greater with unwashed platelets than with washed (glucose-phosphate buffer) platelets, while the reverse was true of net AMP production. An important initial role by adenylate kinase is therefore unlikely in this in vitro system. The net production of both ATP and AMP was much greater in serum derived from non-anticoagulated platelet-rich-plasma and from whole blood than from serum derived from non-anticoagulant platelet-poor-plasma, again emphasizing the importance of platelet enzymes, among others, in the overall reactions observed.

The Effect of Sulfhydrylinhibitors and Glycine Derivatives on Fibrin Polymerization and the Physical Strength of Fibrin in Plasma

SummaryThe influence of p-CMB, iodoacetamide, iodoacetic acid, N-ethylmaleimide, glycerinederivatives and of EDTA on thrombingeneration on fibrinpolymeriza- tion and on the physical strength of clots as expressed by spectrophotometrical and thrombelastographic measurements respectively has been examined.P-CMB and N-ethylmaleimide significantly inhibit the thrombin generation.EDTA retards the initial steps of the fibrinpolymerization and it increases the final turbidity. Its effect is neutralized by calcium and magnesium. The sulfhydrylinhibitors do not inhibit the polymerization neither do the glycinederivatives.To judge from these results does FSF not influence the polymerization but calcium and magnesium do. This statement is verified using FSF deficient plasma.The four SH-inhibitors tested inhibit the development of a normal thromb- elastogram, specially in reducing the mA values. Cysteine, having no effect by itself, reverses the effect of p-CMB and iodoacetamide.p-CMB has a similar effect when added to FSF deficient plasma and the main effect of this SH inhibition is suggested to be on platelet enzymes. p-CMB and iodoacetic acid also reduce the m A values of platelet-free normal plasma possibly by inhibiting FSF.