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.

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.

[Role of the liver in producing heat-stable antifactor Xa]. / Rol' pecheni v produktsii termostabil'nogo antifaktora Xa.

The thermostable antifactor Xa is demonstrable in liver homogenate extracts in an amount exceeding its content in other parenchymatous organs and blood serum. In the course of liver perfusion and incubation of liver sections, the antifactor is demonstrable in the perfused liquid and in the medium used for incubation. Affection of the liver with carbon tetrachloride was attended by the decreased content of the antifactor in serum. The liver seems likely to play an important role in the production of the thermostable antifactor Xa.