[Generation of blood anticoagulant and fibrinolytic activity after intravenous administration of protein C-a in rats]. / Generatsiia antikaguliantnoi i fibrinoliticheskoi aktivnosti krovi pri vnutrivennom vvedenii proteina C-a krysam.

Protein C--vitamin K-dependent protein of the blood coagulation system possessing anticoagulant and fibrinolytic activities was under investigation. Activated partial thromboplastin time was shown to prolong to 214 +/- 8.9% from the first minute after intravenous administration of 0.51 mg per rat bovine protein Ca. After 5 minutes the activity of plasminogen activators increased to 339 +/- 52.8%. Both effects gradually diminished and came back to the starting level within 60-90 minutes. The factor V activity reduced two-fold and didn't return to basal level. We propose that protein Ca reveals its enzymatic activity within first minutes after administration and is blocked then with its inhibitor.

The acceleration of the inhibition of platelet prothrombinase complex by heparin.

The influence of heparin on the inhibition of factor Xa has been studied under conditions where factor Xa is bound to collagen-thrombin-stimulated platelets to form the prothrombinase complex. Unfractionated heparin was found to cause a concentration-dependent acceleration of the inhibition of the platelet prothrombinase complex up to a maximum rate constant of 4.1 X 10(7) M-1 X min-1 at heparin concentrations of 0.2 microM and above. This is equivalent to a 4800-fold acceleration over the rate constant for the inhibition in the absence of heparin, and is 6.8-fold lower than the rate constant for the inhibition of uncomplexed factor Xa in the presence of saturating concentrations of heparin which was determined as 2.8 X 10(8) M-1 X min-1. The effects of three Mr fractions of heparin were also studied. These were a gel-filtered heparin of Mr 15000, a gel-filtered heparin of Mr 6000 and a heparin oligosaccharide (primarily 8-10 monosaccharide units) prepared by nitrous acid depolymerization, each with high affinity for antithrombin III. These fractions all accelerated the rate of the antithrombin III inhibition of the platelet prothrombinase complex, with maximum rate constants of 6.8 X 10(7), 1.4 X 10(7) and 9.8 X 10(6) M-1 X min-1, respectively. On comparison with the effect of these heparin fractions on the rate of inhibition of uncomplexed factor Xa a progressively increasing disparity between the rate of inhibition of uncomplexed and complexed factor Xa was observed, rising from 1.7-fold with the oligosaccharide to 6.8-fold with the unfractionated heparin. A possible mechanism for this differential activity between uncomplexed and complexed factor Xa with the various heparin fractions is discussed in terms of an involvement of heparin binding to factor Xa.

Factor V: a platelet cytoskeletal associated protein.

Platelet cytoskeletons prepared from thrombin activated platelets contain specifically associated Factor Va. We postulate that Factor Va associates with the cytoskeleton through receptors present on the platelet surface. The following evidence supports this hypothesis. (a) Prior secretion of Factor Va is necessary for the association of Factor Va with the cytoskeleton. (b) Reagents that inactivate Factor Va on the surface of the platelet such as EDTA and proteolytic enzymes also inactivate the Factor Va on the cytoskeleton. (c) The platelet Factor Xa binding sites (i.e. Factor Va) are quantitatively retained on the platelet cytoskeleton. (d) Platelet cytoskeletons prepared from platelets that are thought to be deficient in Factor Va binding sites contain 25 percent of Factor Va activity of normal platelet cytoskeletons and platelet cytoskeletons prepared from platelets deficient in Factor Va contain no Factor Va activity but regain control levels of Factor Va only when Factor Va is added to the platelets prior to the preparation of cytoskeletons.

The nature of the binding for prothrombinase at the platelet surface as revealed by lipolytic enzymes.

The nature of the receptor for the prothrombinase complex at the surface of non-activated platelets was investigated by measuring the platelet prothrombin-converting activity wih a chromogenic substrate assay, after treatment of the platelets with various phospholipases or three different proteolytic enzymes. Platelet prothrombin-converting activity only decreased after treatment with those phospholipases which are able to hydrolyse phospholipids in the intact platelet and also have the ability to degrade negatively charged phospholipids, phosphatidylserine and phosphatidylinositol. Those phospholipases which do hydrolyse phospholipids in the intact platelet but have no activity towards phosphatidylserine (and phosphatidylinositol) produce an increase in the platelet prothrombin-converting activity. Proteolytic treatment of platelets with trypsin, chymotrypsin or papain did not result in a decrease of prothrombin-converting activity. It is concluded that negatively charged phosphatidylserine and possibly phosphatidylinositol are involved in the prothrombin-converting activity of non-activated platelets. We could not demonstrate the involvement of platelet membrane proteins in a receptor for the components of the prothrombinase complex.