The class of platelet-specific proteins which displays anti heparin and/or growth stimulating activities includes: high affinity platelet factor 4 (HA-PF-4), low affinity platelet factor 4 (LA-PF-4) and its closely related variant β-thromboglobulin (β-TG), and platelet derived growth factors) (PDGF). In an effort to determine to what extent these proteins and their activities might be broad-based, a comparative study using bovine platelets has been undertaken. Fresh, washed bovine platelets were either thrombin stimulated or freeze-fractured and the ensuing supernatants chromatographed on either heparin-Sepharose (HS) or dextran- sulfate-Sepharose (DSS). Bovine protein which eluted with either 1.0M NaCl (HS) or 1.5M NaCl (DSS) was subsequently gel filtered to homogeneity. Using S2222 chromogenic assays, the bovine protein (9000 dal tons) and human HA-PF-4 (7800 dal tons) each had similar heparin neutral ization equivalence, about 40 U/mg. Using 3T3 cells, growth promoting activity was not found to be associated with the purified bovine HA- PF-4. Although bovine HA-PF-4 has 15 additional residues from the amino terminal end of human HA-PF-4, the acidic, neutral and basic regions have remained similar. HSand DSS fractions from thrombin released platelets eluted with 0.5M NaCl and gel filtered yielded a homogeneous protein of molecular weight 12,000 dal tons. This product has a heparin neutralization activity of approximately 2 U/mg; cellulose acetate electrophoresis of the bovine protein migrated into the gammaglobulin region, and we have consequently referred to it as bovine LA-PF-4. The bovine LA-PF-4 protein had 3T3 growth stimulating activity at least comparable to that of a DSS intermediate salt eluted fraction. The amino terminal 12 residues of bovine LA-PF-4 are not similar to human LA-PF-4 or β-TG. We conclude that these bovine anti heparin proteins are functionally homologous to their human counterparts. We are currently completing the sequence of bovine HA-PF-4 in order to establish the structure-function relationship of the heparin binding domains of these proteins.
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