Boguslaw Rucinski

Interaction of platelet factor 4 with human platelets.

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.

Batroxostatin, an Arg-Gly-Asp-containing peptide from Bothrops atrox, is a potent inhibitor of platelet aggregation and cell interaction with fibronectin

A potent inhibitor of platelet aggregation and cell adhesion was isolated from the venom of Bothrops atrox. This peptide, referred to as batroxostatin, was composed of 71 amino acids and showed a high degree of homology with other snake venom peptides including trigramin, albolabrin, elegantin and applagin: all 12 cysteines and the RGD sequence (standard one-letter amino acid codes) aligned in the same position. Compared on a molar basis, the anti-platelet aggregation activity of batroxostatin was about 1000-times higher than that of RGDS. In addition, batroxostatin was about 400-times more potent than GRGDS at inhibiting melanoma cell adhesion to fibronectin. Batroxostatin covalently attached to plastic promoted adhesion of melanoma cells. The anti-GP140 antibody, recognizing beta 1 integrins, completely inhibited adhesion of mouse melanoma cells to batroxostatin. This observation, in addition to the inhibitory effect of batroxostatin on the adhesion of chick fibroblasts to fibronectin, suggests that batroxostatin interacts with integrins from both the beta 1 and beta 3 subfamilies.

Binding of Dipyridamole to Human Platelets and to α1 Acid Glycoprotein and its Significance for the Inhibition of Adenosine Uptake

The interactions of dipyridamole with alpha(1) acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. Binding studies by equilibrium gel filtration suggested that 1 mol of dipyridamole binds per mol of alpha(1) acid glycoprotein with a dissociation constant of 1.6 muM. Platelets contain two populations of binding sites, one with high and another with lower affinity for the drug. The binding of dipyridamole to the high-affinity sites follows a Michaelis-Menten binding pattern with a dissociation constant of 0.04 muM. Approximately 2 x 10(4) dipyridamole molecules are bound at the high-affinity sites of each platelet. The lower affinity sites bind the drug with a dissociation constant of 4 muM. In the presence of alpha(1) acid glycoprotein of plasma, the binding of dipyridamole to human platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1,000-fold by purified alpha(1) acid glycoprotein. The binding of dipyridamole to human platelets was found to be essential for its inhibition of adenosine uptake by platelets. Dipyridamole decreases the incorporation of [(14)C]adenosine radioactivity in platelet nucleotides and reduces the [(14)C]-ATP to [(14)C]ADP ratio. Purified alpha(1) acid glycoprotein reverses these effects of dipyridamole on adenosine metabolism of platelets in a concentration-dependent manner. An equilibrium of dipyridamole binding to alpha(1) acid glycoprotein and to platelets is proposed.

Subcellular localization of human platelet antiheparin proteins

Abstract The distribution of two platelet antiheparin proteins, platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4) measured by radioimmunoassays and of antiheparin activity measured by a chromogenic assay, was determined in human platelet granule fractions separated by sucrose density centrifugation. Subcellular fractions were characterized by the levels of serotonin (dense granule marker) and β-N-acetylglucosaminidase (lysosomal enzyme marker). PF4 and LA-PF4 antigens and antiheparin activity showed an identical distribution pattern with the highest specific activities occurring in a fraction lighter than that containing serotonin. The fraction with the highest specific activity of β-N-acetylglucosaminidase was observed at a lighter sucrose density than the fraction most enriched in platelet antiheparin proteins. These results indicate that PF4, LA-PF4 and heparin-neutralizing activity are located in the same granule fraction (α-granules) which is different from both the serotonin and the β-N-acetylglucosaminidase-containing fractions. The PF4 and LA-PF4 amounted to 13% of the total proteins of α-granules as determined by radioimmunoassay and SDS polyacrylamide gel densitometric scanning.

Platelet antiheparin proteins and antithrombin III interact with different binding sites on heparin molecule

Heparin acts as an anticoagulant by binding to antithrombin III and by accelerating the rate at which this protein inactivates thrombin and other serine proteases of the intrinsic clotting system [ 11. Purified commercial preparations of heparin contain two species of the molecules that can be separated on the basis of their affinity to antithrombin III [2-41. The high affinity heparin (HA-heparin) binds to antithrombin III in a 1 :l stoichometry withK,8 X lo7 M [5]. Low-affinity heparin (LA-heparin) presumably binds to the same site of antithrombin III with an association constant 100-1000 times lower than that determined for the antithrombin III-HA-heparin binding [6]. The anticoagulant activity of LA-heparin is lo-100 times lower than that of HA-heparin, the ratio varying with the assay system used [7]. There is a good evidence that lysine may function as a heparin-binding site on antithrombin III molecule [ I]. It was suggested [8] that a specific tetrasaccharide with a N-sulfated glucosamine at its reducing end and equivalent amounts of glucuronic and iduronic acid is a specific structural requirement for the interaction of heparin with antithrombin III. More recent data indicate that non-sulfated L-iduronic acid represents a structural feature that is essential for the anticoagulant activity of heparin (U.L., Backstrom, Hook, Thunberg, Fransson and Linker, unpublished data). Stimulated platelets secrete two heparin binding proteins: platelet factor 4 (PF,) and low affinity platelet factor 4 (LA-PF4) [9]. Platelet factor 4 is one of the most potent agents neutralizing the anti-

Human platelet basic protein: Its relation to low affinity platelet factor 4 and β-thromboglobulin

Human beta-thromboglobulin, low affinity platelet factor 4 and platelet basic protein have been purified to homogeneity from the material released by thrombin-stimulated platelets. Purification steps included isoelectric focusing and heparin-agarose chromatography. Antibodies against each of these proteins have been raised in rabbits. Antigenic identity of the proteins has been demonstrated in radioimmunoassay using 125I-labelled platelet basic protein or 125I-labelled low affinity platelet factor 4 and a variety of antibodies. The molecular weight of platelet basic protein estimated by gel filtration in 6 M guanidine hydrochloride using Sepharose 6B corresponded to approx. 10 000 daltons, slightly higher than that of beta-thromboglobulin (8851 daltons) and low affinity platelet factor 4 (9278 daltons). These findings raise the possibility that the formation of low affinity platelet factor 4 beta-thromboglobulin may be a consequence of the action of proteolytic enzymes on platelet basic protein.

Partial purification and characterization of porcine platelet-derived growth factor (PDGF).

Platelet derived growth factor (PDGF) has been partially purified from porcine platelets. Purification steps included heparin-agarose chromatography of the material released by thrombin-stimulated washed porcine platelets and Blue-Sepharose chromatography. Preparative isoelectric focusing showed that isoelectric point of porcine PDGF is at pH 10.0-11.0 and elution experiments from sodium dodecyl sulfate (SDS) polyacrylamide gels indicated that its molecular weight is close to 30 kD. The immunoglobulin fraction prepared from anti-human PDGF serum inhibited the mitogenic activity of porcine PDGF. These experiments suggest a homology of porcine and human PDGF. Porcine platelet factor 4 and porcine platelet basic protein were devoid of significant mitogenic activity.

The effect of dipyridamole on adenosine uptake by platelets ex vivo.

Abstract The inhibition of adenosine uptake by human platelets in the presence of dipyridamole was measured under ex vivo and in vitro conditions. The uptake of [14C]-adenosine by platelets from 12 human volunteers given various dosages of dipyridamole (0.48 to 3.6 mg/kg body weight) correlated with the concentration of the drug in the plasma. On the average, the concentrations of dipyridamole required to produce 50% inhibition of adenosine uptake under in vitro and ex vivo conditions were 0.22 μM and 0.17 μM, respectively. The inhibition of adenosine uptake by platelets of rabbits injected with dipyridamole also correlated with the dosage given. It is suggested that the pharmacological action of dipyridamole on the platelets can be monitored by measuring dipyridamole level in plasma and 14C adenosine uptake by the platelets.

Uptake and processing of human platelet factor 4 by hepatocytes

Abstract We previously demonstrated rapid clearance of human platelet factor 4 (PF4) from rabbit and rat blood, its accumulation in the liver, and elimination of PF4 degradation products in urine. The purpose of the present experiments was to characterize interaction of PF4 with cultured rat hepatocytes. 125I-PF4 was taken up by hepatocytes reaching maximum at 180 min. The association of 125I-PF4 with hepatocytes was two times greater at 37°C than at 4°C. At 37°C degradation of 125I-PF4 by hepatocytes was also observed as indicated by the increase of 125I-PF4 radioactivity soluble in 6% trichloroacetic acid. By contrast, no uptake of 125I-β-throm-boglobulin antigen was observed. Autoradiography demonstrated that short incubation (5-20 min) of 125I-PF4 with hepatocytes results in the association of 125I-radioactivity with cell membranes while after longer incubation (60 min) radioactivity was also localized in the endosomes. Heparin inhibited binding and uptake of 125I-PF4 radioactivity by hepatocytes. We propose that part of PF4 released in the circulating blood by activated platelets is bound to the surface of hepatocytes and that it is further processed by these cells.

[3] Isolation and characterization of porcine platelets

Publisher Summary This chapter discusses the procedures of isolation and characterization of porcine platelets. In the procedures discussed in the chapter, porcine blood was collected and sedimented in 20-liter containers for 2 to 3 hours at a gravitational force. Supernatant was siphoned off and centrifuged in 500-mL polypropylene plastic containers at about 200 g for 15 minutes. Approximately, 2.5–3.0 mL of packed platelets was gently suspended into 40 mL of washing fluid calcium-free Tyrodes buffer (A) or sodium chloride citrate (B). The optimal conditions for the separation of human and porcine platelets differ. Differential centrifugation followed by resuspension in Ca 2+ , Mg 2+ , Tyrodes buffer with apyrase at pH 7.35 is the most suitable method for the large-scale separation of fresh human platelets. The usage of slightly acidic washing fluid A or B gave better results with porcine platelets. Porcine platelets require high concentrations of salt (osmolarity 340) for their stability as compared to human platelets.