Biochemical and biophysical aspects of human platelet adhesion to collagen fibers

Abstract

A B S T R A C T A method has been developed for measuring the adhesion of platelets to purified collagen fibers obtained from bovine tendon. This method differs from others in that: (a) platelet adhesion is measured in the absence of platelet aggregation; (b) platelet-rich plasma collected in ACD (acid citrate dextrose) or EDTA, or washed platelets can be employed; (c) adherent platelets are enumerated directly; (d) erythrocytes and leukocytes do not adhere.Washed platelets suspended in human Ringer solution exhibit negligible adhesion (at the platelet concentrations employed) in contrast to washed platelets suspended in plasma. Addition of purified human fibrinogen (95% clottable, 2-4 mg/ml) to human Ringer solution completely restores the ability of washed platelets to adhere to collagen fibers. Albumin (fatty acid free, 50 mg/ml) is also capable of restoring adhesion. Albumin and seven other proteins at concentrations of 5-10 mg/ml, with varying molecular weights, isoelectric points, and frictional coefficients are incapable of supporting the adhesion of washed platelets. The proteins tested were human globulin, hexokinase, hemoglobin, cytochrome-C, insulin, thyroglobulin, and muramidase. Platelet adhesion is proportional to both platelet concentration and fibrinogen concentration, but is independent of temperature or glycogen stores.Modification of fibrinogen by acylation of amino groups or removal of sialic acid has no effect on its ability to support platelet adhesion. Degradation of fibrinogen with purified plasmin results in decreased support of platelet adhesion. This accompanied formation of early breakdown products with clottability ranging from 84-0%. Formation of fibrinogen degradation products was monitored by SDS-polyacrylamide gel electrophoresis of the corresponding fibrins after reduction of Received for publication 21 January 1971 and in revised form 17 February 1971. disulfide bonds (a method capable of distinguishing a-, P-and gamma-chains). Decreased support of platelet adhesion is associated with the disappearance of intact a-chains and early modification of the a-chains.Purified proteinpolysaccharide macromolecules obtained from bovine nasal and humeral cartilage, and from nucleosus pulposus are as effective as fibrinogen on a weight basis and ten to thirty times more effective on a molar basis in supporting platelet adhesion. The purified mucopolysaccharide side chains: chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan-sulfate are incapable of supporting platelet adhesion. INTRODUCTIONAlthough considerable information has been accumulated regarding the biochemistry and physiology of platelet-plug formation (1-7), little is known about initiation of platelet plug formation, namely platelet adhesion. Early work, employing light microscopy, (8)(9)(10)

Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo.

“…Triton-solubilized platelet extracts were prepared from outdated Tisch Hospital Blood Bank platelets. Centrifuged platelets were washed in a modified human Ringer solution (20) containing the protease inhibitors: 10 mM EDTA, 10 mM benzamidine, 100 Mg/ml soybean trypsin inhibitor, and 0.1 mM PMSF, and solubilized in the same solution to which 1% Triton X-100 has been added. This extract was sonicated at 4°C for 20-30 s at 60 W, and then allowed to stand for 1 h at 4°C.…”

Heterogeneity of human platelets

“…Human platelets were obtained, collected, and processed as described previously (4). The washed platelets were suspended in a human Ringer solution (8) and separated into heavy and light platelet populations as described previously (4).…”

Anti-human immunodeficiency virus type 1 antibody complexes on platelets of seropositive thrombocytopenic homosexuals and narcotic addicts.

“…Platelet-rich plasma was prepared from EDTA/anticoagulated blood as described (9) and was either further processed by centrifugation and washing as described (9) for elution and binding experiments or placed on a Sepharose 2B gel-filtration column (Pharmacia) for DNA extraction. The gel-filtration procedure was employed to prepare platelets entirely free of lymphocytes.…”

“…Platelet eluates were prepared by acid elution (16). Briefly, 1 x 108 washed platelets (9) were suspended in 200 ,ul of a modified human Ringer's solution (9) and acidified by addition of 100 ,u of 150 mM H3PO4/100 mM NaCl/1.5% bovine serum albumin, pH 2.8, for 10 min, with shaking at 37°C. In some experiments, the platelet suspension was frozen and thawed three times with dry ice/ethanol prior to acidification with similar results.…”