Gwendolyn J. Stewart

Neutrophils and deep venous thrombosis.

A case can be made for the participation of polymorphonuclears (PMN) in the initiation and propagation of venous thrombosis. In animal models leukocytes adhered to areas of veins that serve as sites for initiation of thrombi in patients. In addition, PMN are found in white layers of thrombin where they may interact with platelets to attract more of each. This would add bulk and promote coagulation so that red layers are formed. Lidocaine and one of its derivatives inhibited leukocyte adhesion to veins in dogs and lidocaine reduced the incidence of deep venous thrombosis (DVT) in patients after hip replacement, suggesting but not proving that inhibition of PMN adhesion might have contributed. A new approach for preventing PMN contribution to DVT is suggested by recent studies which identified three families of adhesive receptors (integrins, intercellular adhesion molecules and selectins) on endothelium, leukocytes and platelets. Monoclonal antibodies against beta 2-integrins on leukocytes reduced leukocyte adhesion, emigration and PMN-dependent tissue injury in infection, inflammation and ischemia-reperfusion injury in animals. Selectins bind to specific carbohydrate ligands containing sialylated Lewis X, suggesting that relatively small analogues might inhibit PMN adhesion. Both platelets and PMN adhere to polymerizing fibrin through undefined mechanisms. Inhibition of this process might inhibit the buildup of white layers of thrombi.

Hereditary Giant Platelet Syndrome

Summary The platelets from two related patients with the hereditary giant platelet syndrome were examined. They were larger than normal but otherwise ultra‐structurally normal; they contained increased storage pools of adenine nucleotides and heparin‐neutralizing activity and took up serotonin at an increased rate. They aggregated normally with ADP and collagen but failed to aggregate with bovine factor VIII and Ristocetin. Some change in shape occurred with ADP, and the reduction in adenylate energy change after addition of ADP to platelet‐rich plasma was smaller than normal.

The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin.

Flavoridin and echistatin, isolated from the venom of Trimeresurus flavoviridis and Echis carinatus, respectively, belong to the disintegrin family of integrin β1 and β3 inhibitors of low molecular weight RGD‐containing, cysteine‐rich peptides. Since disulfide bonds are critical for expression of biological activity, we sought to determine their location in these two proteins. In flavoridin, direct evidence for the existence of linkage between Cys4‐Cys10 and between Cys45 and Cys64 was obtained by analysis of proteolytic products, and indirect evidence suggests links between Cys13‐Cys14 and Cys13‐Cys14. In echistatin, links between Cys14‐Cys37 and Cys20‐Cys34 were identified by direct chemical analysis.

Platelet factor 4 efficiently reverses heparin anticoagulation in the rat without adverse effects of heparin-protamine complexes.

BackgroundIt has been observed that the reversal of heparin anticoagulation in humans by protamine sulfate (PS) results in various adverse reactions including leukopenia, thrombocytopenia, activation of complement, increased vascular permeability, systemic hypotension, pulmonary vasoconstriction, and pulmonary edema. The purpose of this study was to compare the efficacy and effects of native platelet factor 4 (PF4) and recombinant platelet factor 4 (rPF4) with those of PS in heparin neutralization in vivo, using a rat model. Methods and ResultsSprague-Dawley rats were anesthetized with sodium pentobarbital, and the right femoral vein and carotid artery were cannulated. For determination of activated partial thromboplastin time, platelet count, white blood cell count, and complement titer, arterial blood samples were taken before and immediately after heparin (10 units/100 g) infusion and at several time points after the infusion of the neutralizing agent (PS, 0.1 mg/100 g; PF4, 0.5 mg/100 g). In separate groups of animals, mean arterial blood pressure was monitored throughout identical protocols and the lungs were prepared for histological examination. The anticoagulant activity of heparin was effectively reversed by all of the neutralizing agents (PS, PF4, and rPF4). Platelet count (48% of initial), white blood cell count (52% of initial), complement titer (60% of initial), and mean arterial pressure (20% decrease) decreased significantly in heparinized animals receiving PS but not in those receiving PF4 or rPF4. Lung interstitium appeared normal when heparin was followed by PF4; however, interstitial edema and hemorrhage were observed with heparin-PS. ConclusionsThese results suggest that PF4 efficiently reverses heparin anticoagulation in the rat without the adverse effects of heparin-protamine complexes. Therefore, rPF4 may be an appropriate substitute for PS in patients undergoing cardiovascular surgery and other procedures that require heparin anticoagulation.

Formation of highly ordered polymers from fibrinogen and fibrin degradation products

Abstract Products of limited plasmin digestion of fibrinogen (FDP) and of fibrin (fdp) retained the ability to form highly organized polymers with protamine sulphate while products of extensive digestion lost this ability. Electron microscope studies showed that highly organized polymers were formed only in samples of FDP or fdp which contained Fragment X. Strong evidence indicated that there are two varieties of Fragment X, one with fibrinopeptides (Xfp) and one devoid of fibrinopeptides (Xo). The significance of fibrinopeptides on the behavior of the fragment was indicated by the difference in mechanisms by which polymers seem to be formed. Fragment Xfp (contained in FDP or purified) reacted with protamine sulphate but not ethanol to form rather small lancet-shaped polymers with a periodicity of 225–240 A without branching. Fragment Xo (contained in fdp) reacted with protamine sulphate and with ethanol to form an extensive net which was indistinguishable from thrombin induced fibrin in extent, branching and periodicity. This likely resulted from dissociation of complexes of Xo with other degradation products Y or D, followed by the spontaneous polymerization of Xo. Purified Fragment Xfp formed a similar net after conversion to Xo by thrombin. After pretreatment with thrombin (to remove fibrinopeptides from Xfp), FDP formed an extensive net with both protamine sulphate and ethanol, probably by a mechanism similar to that described for dissociation of complexes in fdp. Maximal protein precipitation from fibrinogen and FDP required 5–10 times as much protamine sulphate as was required for similar precipitation from fdp and FDP pretreated with thrombin. As digestion progressed, the amount of precipitable protein decreased until only a small amount remained. The formation of highly ordered fibrin-like polymers in fibrin or fibrinogen digests appeared to result from (a) the direct action of protamine sulphate on fibrinogen and Fragment Xfp; (b) the dissociation by ethanol or protamine sulphate of complexes of Fragments Xo with Y or D followed by the subsequent polymerization of Fragment Xo. The latter mechanism is probably responsible for the paracoagulation reaction.

Intraoperative venous dilation and subsequent development of deep vein thrombosis in patients undergoing total hip or knee replacement.

This patient study was based on the observation of characteristic intimal lesions in jugular and femoral veins removed from dogs a few hours following total hip replacement. The lesions, small localized intimal tears, suggested that smooth muscle and connective tissue, might have dilated beyond the ability of intima to accommodate. Intraoperative venous dilation correlated with the incidence of intimal lesions. It was postulated that surgical trauma resulted in circulating vasoactive substances which caused venous dilation and that dilation of smooth muscle and connective tissue beyond the yield point of intima resulted in intimal rupture. Similar intraoperative dilation and lesions, in patients might predispose to development of deep vein thrombosis (DVT). Total hip (THR) and total knee (TKR) replacement patients were selected for study because: (a) of the high incidence of DVT and (b) blood circulation is present in THR but not in TKR patients during operation. Ultrasound was used to monitor cephalic vein diameter during the perioperative period. Development of DVT postoperatively was compared with intraoperative venous dilation. In THR patients, intraoperative venous dilation ranged from 6%-56%. One of nine patients with dilation less than or equal to 17% developed DVT while 12 patients with dilation of greater than or equal to 22% developed DVT, giving a correct prediction of 95%. Of four patients in the intermediate range (19%, 20%), two developed DVT and two did not. The sharp demarcation was to be expected because of abrupt rupture of viscoelastic material when the critical point of elongation is exceeded.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of three calcium antagonists on platelet secretion and metabolism.

Abstract Three compounds, 8-( N , N -diethylamino-octyl 3,4,5-trimethoxybenzoate, HCl (TMB-8), 2-propyl-3-dimethylamino-5,6-methylenedioxyindene HCl (2-PIA), and chlortetracycline, were investigated to determine whether their effects on washed human platelets were compatible with a suggested role as calcium antagonists. TMB-8 had little effect on levels of metabolic ATP and IMP, whereas chlortetracycline caused a decrease in metabolic ATP and an increase in IMP; both compounds inhibited thrombin-induced secretion and changed the platelets to spheres. At concentrations up to 0.5 mM, 2-PIA caused a decrease in ATP and an increase in IMP, an induction of secretion, and a centralization of electron-dense material in the platelets, all changes which suggest induction of secretion. The ultrastructural, functional and metabolic effects of TMB-8 and the type of interference by the drug with the effects of thrombin and 2-PIA on the same variables suggest that TMB-8 is mainly a membrane-active drug. Chlortetracycline on the other hand caused changes in platelet metabolism, ultrastructure, and function which, at least partly, indicate an effect on intracellular mechanisms. Both TMB-8 and 2-PIA, and to lesser degree chlortetracycline, caused loss of cytoplasmic nucleotides from the platelets.

Method of predicting the occurrence of deep vein thrombosis by non-invasive measurement of vessel diameter

A method of predicting the occurrence of deep vein thrombosis after a surgical procedure comprises monitoring changes in the internal diameter of a blood vessel using a non-invasive ultrasound technique. The frequency and magnitude of changes in vessel diameter are used to predict whether deep vein thrombosis will occur.

Isolation characterization and immunological detection of neutrophil activating peptide 2. a proteolytic degradation product of platelet basic protein

Abstract Neutrophil-activating peptide 2 (NAP-2), corresponding to platelet basic protein fragment 25–94, was prepared by chymotryptic digestion of its precursors, low affinity platelet factor 4 or β-hromboglobulin, followed by purification by high performance liquid chromatography. NAP-2 (0.1–1.5 μm) caused the release of human granulocyte elastase from cytochalasin B-treated neutrophils in a dose-dependent manner. In the same system, β-thromboglobulin, human platelet factor 4, S-pyridylethyl NAP-2, and platelet basic protein C-terminal fragment (77–94) were inactive, whereas platelet basic protein fragment 22–89 had low, but significant, activity. Sensitive immunological identification of NAP-2 based on nonequilibrium isoelectric focusing and immunoblotting is described.

Cytoplasmic free calcium concentration in porcine platelets: Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1 X 10(-7) M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thrombin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.