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The fibrinolysis in 6 patients with lupus anticoagulant was studied. The intrinsic fibrinolytic activity by kaolin activated euglobulin with plasminogen free fibrin plate was low level in all patients. Although we could not obtain common results in fibrinolytic factors-plasminogen, α1-AT, α2-M, α2-PI and Cl-inactivator with immunodiffusion method, and in fibrinolytic activity by euglobulin from plasma with ELT and plasminogen rich fibrin plate.In two patients with Budd-Chiari syndrome, the IgG fraction of plasma through Sephadex G 200 had the inhibitory activity of fibrino (geno) lysis on the fibrin plate method and the immunoelectrophoresis, as well as high lupus anticoagulant activity.Those findings suggest that fibrinolysis plays a role on formation of thrombus in patient with lupus anticoagulant.

Arachidonate (AA) stimulated platelet protein kinase C in a dosedependent manner (5-50μg/ml), when protein kinase C was assayed with either histone H-1 or myosin light chain. Arachidic acid and methyl arachidonate did not stimulate protein kinase C. AA also stimulated the endogenous phosphorylation of 40K and 20K proteins in the cytosol (100, 000g sup.) of human platelets. When intact platelets were activated by AA, a biphasic pattern of platelet aggregation and secretion were observed corresponding to the dose of AA, and these responses were associated with the phosphorylation event of 40K and 20K proteins. Platelet activation induced by the low level of AA (0.1-5μg/ml) was inhibited by aspirin, whereas that induced by the high level of AA (10-50μg/ml) was not inhibited. Arachidic acid and methyl arachidonate did the not induce platelet responses. These results suggest that the ability of AA to activate directly protein kinase C may account for the activation of platelets by high level of AA.

In order to elucidate the physiological role of calcium binding protein, calmodulin in platelet aggregation phenomenon, we determined the effect of the calmodulin-interacting agent, which selectively binds to calmodulin and inhibits Ca2+-calmodulin activated enzymes, on the endogenous phosphorylation of myosin light chain from human platelets. An actomyosin enrich fraction contained approximately 50 units of calmodulin per mg protein. N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) as a calmodulin-interacting agent produced dose-dependent inhibition of 32P incorporation into 20, 000 dalton myosin light chain from [γ-32P] ATP in the presence of calcium ions (100μM) with the IC50 value of 75μM. However within the concentration range examined, W-7 was not effective to inhibit the phosphorylation of MLC in the absence of calcium (EGTA 5mM).Moreover, W-7 was able to inhibit in dose-dependent fashion platelet aggregation induced by various aggregating agents such as adenosine diphosphate (ADP), collagen, epinephrine or restocetin with indistinguishable IC50 values to that observed in phosphorylation inhibition. Our results provide pharmocological evidence that calmodulin-mediated system, especially Ca2+-dependent phosphorylation of platelet myosin light chain play an important role and is common process in platelet aggregation phenomenon induced by any aggregating agents.

Polymyxin B (PMB), a cyclic polycationic peptide antibiotic, inhibited dose-dependently human platelet aggregation induced by a high level (45μg/ml) of arachidonate (AA) or eicosadienoic acid (C20: 2), whereas this drug had no effect on a low level (0.45μg/ml) of AA-induced platelet responses. Colistin (polymyxin E), a derivative of PMB, had no inhibitory effect on platelet aggregation induced by a high level of AA or C20: 2. When human platelets were stimulated with AA, phosphorylation of 40K and 20K proteins preceded the aggregation response and platelet response by AA correlated closely with the phosphorylation of these proteins. PMB (100μM) inhibited the phosphorylation of 40K and 20K proteins induced by 45μg/ml AA, but not the phosphorylation event induced by 0.45μg/ml AA. PMB also inhibited dose-dependently [Ca2+]i elevation in response to a high level (20μg/ml) of AA, whereas PMB had no effect on a transient increase in [Ca2+]i induced by a low level (0.45μg/ml) of AA. The present results indicate that PMB has no effect on protein phosphorylation systems in human platelets although the drug has been shown to inhibit protein kinase C in vitro, and suggest that PMB selectively inhibit platelet aggregation induced by a high level of AA, possibly by interacting the lipid matrix of platelet membranes.

Protein kinase C was purified to the apparent homogeneity from a 100, 000×g supernatant fluid of human platelets homogenate, employing the steps of DEAE cellulose, Ultrogel AcA-34 and phenyl-Sepharose CL-4B chromatographies. The purified enzyme appeared as a single protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, having a Mr 82, 000. With histone H-1 as a substrate its activity was 20-fold higher in the presence of phospholipid and Ca2+ than in the presence of phospholipid and EGTA, EGTA or Ca2+ alone. The enzyme has a Km of 8.3μM and a Vmax of 1.5μmol/mg/min for histone H-1. Platelet protein kinase C undergoes an apparent autophosphorylation. The enzyme was most active at 10-20mM mg2+ with histone H-1 as a substrate, wherease with mixed myosin light chains from turkey gizzard the maximum activity was observed at 1mM mg2+. Of several myosin light chains isolated from a variety of sources, the enzyme phosphorylates the 20, 000-dalton light chain of myosin from human platelets and turkey gizzard smooth muscle at a significant rate. The enzyme also phosphorylates the two phosphorylatable light chains of myosin from Limulus. These results suggest that platelet protein kinase C can phosphorylate the regulatory light chain of myosin non-muscle and smooth muscle, in which myosin phosphorylation catalyzed by myosin light chain kinase is thought to control contraction and cytoskeletal related processes.