No abstract

The procoagulant protein F.VIII:C is noncovalently bound to von Willebrand factor (vWF) to give the factor VIII macromolecular complex. New highly purified preparations of isolated human F.VIII:C, devoid of vWF and about 500,000-fold purified, were administered to hemophilia A and von Willebrand disease (vWD) dogs to determine their hemostatic effectiveness and survival in the circulation. Two preparations of F.VIII:C were used: peak 1, with active components of Mr 185,000-280,000, and peak 2, with a single component of Mr 170,000. In hemophilic dogs, with no plasma F.VIII:C but normal vWF, both preparations immediately elevated plasma F.VIII:C to expected levels, promptly stopped induced and spontaneous hemorrhages, and gave sustained plasma levels of F.VIII:C. The isolated F.VIII:C immediately complexed with endogenous vWF in hemophilic plasma and was eliminated exponentially, with a half-life (t1/2) of about 9 hr. Survival of peak 2 F.VIII:C was longer than that of peak 1 material. In contrast, F.VIII:C complexed to vWF in a therapeutic concentrate administered to hemophilic dogs was eliminated biexponentially with first-phase t1/2 of 3.2 hr and second-phase t1/2 of 9 hr. In vWD dogs with no vWF and reduced F.VIII:C levels, the isolated F.VIII:C produced supernormal levels of F.VIII:C without effect on induced bleeding. It was rapidly eliminated from plasma with a t1/2 of about 1 hr, as was the complexed F.VIII:C in the concentrate. These data indicate that isolated F.VIII:C promptly complexes with vWF and in this form is highly effective in controlling hemophilic hemorrhages with good survival in plasma. Without endogenous vWF with which to complex, the F.VIII:C is promptly eliminated.

Oral administration of the DTI ximelagatran resulted in a rapid inhibition of both thrombin generation and platelet activation in a concentration-dependent manner using a human shed blood model. The inhibition of thrombin generation by 60 mg ximelagatran was comparable to that observed with doses of r-hirudin and enoxaparin demonstrated to be effective for the treatment of acute coronary syndromes.

No abstract

Thrombin-induced platelet activation involves cleavage of protease-activated receptors (PARs) 1 and 4, and interaction, via glycoprotein (Gp)Ibalpha, with the platelet GpIb/IX/V complex. This study investigated inhibition of platelet activation by thrombin inhibitors with different modes of action: two reversible direct thrombin inhibitors, melagatran and inogatran; hirudin, a tightly binding direct thrombin inhibitor; and two indirect thrombin inhibitors, heparin and dalteparin. Up-regulation of P-selectin (CD62P) and PAR-1 cleavage was measured in human whole blood, by flow cytometry. The thrombin concentration that induced 50% of maximum (EC50 ) PAR-1 cleavage was 0.028 nmol/l, while that of platelet activation (CD62P) was over two-fold higher (0.64 nmol/l). The EC50 of a PAR-1-independent component, defined as a further activating effect of thrombin on top of the maximum PAR-1-activating peptide (AP) effect, was 3.2 nmol/l. All anticoagulants were concentration-dependent inhibitors of thrombin-induced platelet activation and PAR-1 cleavage, but none inhibited PAR-1-AP or PAR-4-AP induced activation. Melagatran and inogatran were more potent inhibitors of CD62P up-regulation than of PAR-1 cleavage; conversely, hirudin, heparin and dalteparin were more potent inhibitors of PAR-1 cleavage.Thus, reversible direct thrombin inhibitors, such as melagatran, are potent inhibitors of thrombin-induced platelet activation, acting mainly by inhibition of a PAR-1-independent component.

Coronary artery reocclusion after thrombolysis with human recombinant tissue-type plasminogen activator (rt-PA) is related to the short half-life of this agent in plasma. K2P, a mutant of rt-PA lacking the fibronectin fingerlike, epidermal growth factor-like and first kringle domains (amino acids 6 to 173) and having the glycosylation site Asn184 mutagenized to Gln, has been produced in Chinese hamster ovary cells. In this study we compared the thrombolytic effect of K2P and rt-PA in dogs with electrically induced coronary artery thrombosis. Both agents were given intravenously in equimolar amounts over 20 min after the occlusive thrombus was stable for 30 min; dogs were monitored for 1 h after reperfusion if flow occurred. Coronary blood flow was restored by rt-PA in 6 (60%) of 10 dogs. The restored flow lasted for 49 +/- 12 min and mean flow at 60 min from the start of reperfusion was 7 +/- 3 ml/min. The reocclusion rate was 50% (three of six dogs). Flow was restored in five (100%) of five dogs by K2P. The restored blood flow lasted during the entire 1-h observation period in all but one dog and mean flow at 60 min was 49 +/- 16 ml/min (p less than 0.02 vs. flow in rt-PA-treated dogs). Restored coronary blood flow showed marked cyclic flow variations in rt-PA-treated but not in K2P-treated dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

1 The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y 12 and P2Y 1 inhibition and P2Y 12 and thrombin inhibition, on ADP-and thrombin-induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both Ga q and Ga i signalling. 2 Blood from healthy volunteers was preincubated with AR-C69931MX, a reversible P2Y 12 antagonist; MRS2179, a reversible P2Y 1 antagonist; or melagatran, a direct reversible thrombin inhibitor; alone or in various combinations prior to activation with ADP or thrombin. Platelet function in whole blood was assessed by flow cytometry using the antibody PAC-1 to estimate the expression of active a IIb b 3 (the fibrinogen receptor GPIIb/IIIa). A synergistic effect was evaluated by comparing the concentrations in the different combinations with those of corresponding equipotent concentrations of each single inhibitor alone. The equipotent single concentrations were experimentally obtained from concentration response curves performed in parallel. 3 A synergistic effect regarding inhibition of ADP-induced platelet activation (10 mM) was obtained with different combinations of AR-C69931MX and MRS2179. 4 Inhibition of thrombin-induced platelet activation (2 nM) with combinations of AR-C69931MX and the thrombin inhibitor melagatran did also result in a strong synergistic effect. 5 To our knowledge, this is the first time that data supporting a synergistic effect has been published for the inhibitor combinations described. 6 Whether this synergistic effect in vitro also results in an improved antithrombotic effect in vivo with or without an increased risk of bleeding remains to be studied in well-conducted clinical studies.

SummaryA sensitive thrombosis model with a high reproducibility was developed in the rat, utilizing stasis of the caval vein and a standardized surgical trauma as the only thrombogenic stimuli. Since no procoagulant substances were used, the results of the present study might be relevant in a clinical situation. The antithrombotic effect of two recently synthesized low-molecular-weight thrombin inhibitors have been compared to dalteparin, (Fragmin) a low-molecular-weight heparin fragment. Each compound was studied at 8 different doses with 10 rats in each group. On a gravimetric basis, the thrombin inhibitor melagatran was twice as potent as dalteparin (ED50 16 and 33 µ/kg per h, respectively). The second thrombin inhibitor, inogatran, had an intermediate effect, with an ED50 of 24 µLg/kg per h. No differences in antithrombotic effect were, however, found when the compounds were compared at anticoagulant equivalent doses (same APTT prolongation). A 50% reduction in the mean thrombus weight was obtained when APTT was prolonged to 1.2 to 1.3 times the pretreatment value.