Kinetics of thrombopoiesis.

Measurements of megakaryocyte number, volume, nuclear number, and cytoplasmic granulation were compared in animals with induced thrombocytosis, normal platelet counts, and induced thrombocytopenia for 4- and 10-day periods. Thrombopoiesis, as measured by megakaryocytic mass and (35)S incorporation into platelets, appeared to be regulated by the demand for circulating platelets. In addition, two mechanisms were operative in the alteration of thrombopoiesis. The first mechanism involved the regulation of endomitosis in the immature megakaryocytes which in turn determined the subsequent volume of platelet-producing cytoplasm. The second mechanism regulated the number of megakaryocytes formed from the precursor, "stem cell," compartment.

Magakaryocyte quantitation.

A method is described for the quantitation of megakaryocytes. In this technic bone marrow sections obtained at the time of maximal marrow uptake of previously injected plasma bound (59)Fe are counted for radioactivity as well as the number of megakaryocytes and nucleated red cells. We calculated the total megakaryocytes by relating the ratio of megakaryocyte: radioactivity with the total marrow (59)Fe activity at the time of sampling. A multiple counting correction factor which is related to cell diameter and section thickness is required for quantitation of cell numbers from section material. The normal megakaryocyte number for the rat is 11.0 x 10(6)/kg, and 6.1 x 10(6)/kg for man.

Experimental arterial thromboembolism in baboons. Mechanism, quantitation, and pharmacologic prevention.

A quantitative primate model of arterial thromboembolism has been characterized with respect to mechanism and usefulness in evaluating modifying variables. The model involved the kinetic measurements of (51)Cr-platelets and (125)I-fibrinogen consumption by femoral arteriovenous cannulae in chaired baboons. Cannula platelet consumption correlated directly with exposed cannular area for irradiated Silastic and polyurethane (correlation coefficients of 0.940 and 0.901, respectively; P < 0.001) and remained steady state for months. Nonirradiated Silastic was only minimally reactive with platelets. Despite increased rates of platelet consumption circulating fibrinogen was not measurably destroyed by any of the cannulae tested. Cannula platelet consumption was independent of cannula flow rate, platelet count, heparin anti-coagulation, and ancrod defibrinogenation.(111)In-platelet imaging of irradiated Silastic cannulae demonstrated luminal accumulation and subsequent embolization of irregular platelet masses. When irradiated Silastic cannulae were inserted as extension segments in the renal arteries of four animals the glomerular vessels became progressively occluded with nonfibrin-containing platelet thromboemboli. Nonirradiated Silastic cannulae in control arteries produced no significant vascular occlusion. Because the survival of platelets from animals with consumptive cannulae was not shortened in normal recipient animals we concluded that platelets were either irreversibly removed through thromboembolic consumption or unaffected in their viability. Oral administration of dipyridamole and sulfinpyrazone decreased cannula platelet consumption in a dose-dependent manner with complete interruption at 20 and 250 mumol/kg body wt per d (in three divided doses), respectively, whereas oral acetylsalicylic acid (10-330 mumol/kg per d) had no measurable effect on cannula platelet consumption. We conclude that this primate model simulates arterial thrombotic processes in man and that this model is suitable for the in vivo evaluation of biomaterials and of drugs that modify platelet behavior.

Thrombokinetics in man.

Platelet production, distribution, and destruction have been quantitated in normal man and in selected patients with platelet disorders. In most instances, total production as calculated from the megakaryocyte mass agreed with production estimated from platelet turnover. In patients with megaloblastosis, a discrepancy between these two measurements indicated the presence of ineffective thrombopoiesis. Thrombopoiesis was regulated by (a) alterations in megakaryocyte number, and (b) changes in megakaryocyte volume (produced by changes in endomitosis). The volume-endomitosis changes were closely related to the peripheral platelet count and were a useful indicator of thrombopoietic stimulus. Thrombocytopenic disorders have been classified on the basis of the disturbed physiology into disorders of (a) production (hypoproliferative or ineffective), (b) distribution (splenic pooling), or (c) destruction (immune or consumptive). Less than a twofold increase in platelet production in the presence of significant thrombocytopenia was taken to represent impaired proliferation. Thrombocytosis was classified as reactive or autonomous. Reactive thrombocytosis was consistently associated with a mean megakaryocyte volume and endomitosis less than normal but appropriate for the elevated circulating platelet count. In contrast, the average megakaryocyte volume and nuclear number were always greater than normal in thrombocythemia findings indicating autonomy.

Effects of platelet-modifying drugs on arterial thromboembolism in baboons. Aspirin potentiates the antithrombotic actions of dipyridamole and sulfinpyrazone by mechanism(s) independent of platelet cyclooxygenase inhibition.

To resolve questions of drug actions, efficacy, and interactions for platelet-modifying agents used clinically, we have compared the relative capacities and mechanisms of aspirin, dipyridamole, sulfinpyrazone, and dazoxiben to prevent arterial thromboembolism in a baboon model. In 136 studies the agents were given twice daily by oral administration both singly and in combination. The antithrombotic efficacy of a given therapy was determined by its capacity to interrupt steady-state platelet utilization induced by thrombogenic arteriovenous cannulae. When given alone, dipyridamole and sulfinpyrazone reduced the rate at which platelets were utilized by thrombus formation in a dose-dependent manner with essentially complete interruption by dipyridamole at 10 mg/kg per d. In contrast, neither aspirin (2-100 mg/kg per d) nor dazoxiben (20-100 mg/kg per d) decreased cannula platelet consumption detectably despite the striking reduction in the capacity of platelets to produce thromboxane B2. However, aspirin, but not dazoxiben, potentiated the antithrombotic effects of dipyridamole and sulfinpyrazone in a dose-dependent fashion without changing the pharmacokinetics for any of the agents. Complete potentiation required aspirin at 20 mg/kg per d to be given with each dose of dipyridamole. Because dazoxiben's blockade of platelet thromboxane A2 production was not associated with antithrombotic potentiation, and because complete potentiation by aspirin required a dose that fully inhibited vascular production of prostaglandin I2 (PGI2), we conclude that aspirin's potentiating effect on dipyridamole is independent of PGI2 production or inhibition of thromboxane A2 formation. In addition, because frequent repeated and synchronous dosing of aspirin was necessary, aspirin's potentiating effects appear to be produced by mechanism(s) unrelated to its potent, irreversible inhibition of platelet cyclooxygenase.

Homocystine-induced arteriosclerosis. The role of endothelial cell injury and platelet response in its genesis.

The atherogenic mechanism of homocystinemia has been defined by measuring endothelial cell loss and regeneration, platelet consumption, and intimal lesion formation in a primate model. Three groups of baboons were studied: (a) 8 control animals; (b) 15 animals after 3 mo of continuous homocystinemia; and (c) 11 animals after 3 mo of combined homocystinemia and oral treatment with dipyridamole. Experimental homocystinemia caused patchy endothelial desquamation comprising about 10% of the aortic surface despite a 25-fold increase in endothelial cell regeneration. Neither endothelial cell loss nor regeneration was changed significantly by dipyridamole. Homocystine-induced vascular deendothelialization produced a threefold increase in platelet consumption that was interrupted by dipyridamole inhibition of platelet function. All homocystinemic animals developed typical arteriosclerotic or preatherosclerotic intimal lesions composed of proliferating smooth muscle cells averaging 10-15 cell layers surrounded by large amounts of collagen, elastic fibers, glycosaminoglycans, and sometimes lipid. Intimal lesion formation was prevented by dipyridamole therapy. We conclude that homocystine-induced endothelial cell injury resulted in arteriosclerosis through platelet-mediated intimal proliferation of smooth muscle cells that can be prevented by drug-induced platelet dysfunction.

Neutrophil kinetics in the dog.

The production of neutrophils in dogs has been estimated from the number of postmitotic neutrophils in the marrow and the transit time of a [3H]-thymidine pulse. The number of postmitotic neutrophils was derived from the erythron iron turnover measurement of erythroid number and the neutrophil-erythroid ratio in bone marrow sections. The mean value for marrow postmitotic neutrophils in dogs was 5.61 plus or minus 0.56 times 10-9 cells/kg. The mean transit time of these neutrophils was calculated to be 82.1 h. A marrow production of 1.65 times 10-9 neutrophils/kg/day was calculated from these data. The turnover of circulating neutrophils was measured by [3H]thymidine and [32P]diisopropylphospho-fluoridate (DF32P) labeling of blood neutrophils. [3H]-Thymidine labeling gave a calculated recovery of 65 per cent, a t1/2 disappearance time of 6.7 h, and a calculated turnover of 1.66 times 10-9 cells/kg/day. Corresponding results with DF32P tagging were 51 per cent, 5.4 h, and 2.89 times 10-9 cells/kg/day. The discrepancy between these two tags persisted in doubly tagged cells and was considered to be due to elution of DF32P.

Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cells.

The effect of thrombin on the release of tissue plasminogen activator from endothelial cells was studied in primary cultures of human umbilical vein endothelial cells. Tissue plasminogen activator concentration in conditioned medium was measured by a two-site radioimmunometric assay. The addition of increasing concentrations (0.01 to 10 U/ml) of thrombin to confluent cultures produced a saturable, dose-dependent increase in the rate of release of tissue plasminogen activator. A sixfold increase in tissue plasminogen activator concentration (from 2 to 12 ng/ml) occurred after the addition of 1 U/ml thrombin (8 X 10(-9) M) to cultures containing 5 X 10(4) cells/cm2. Enhanced release was not observed until 6 h after thrombin addition, reached a maximum rate of 1.3 ng/ml per h between 8 and 16 h, and then declined to 0.52 ng/ml per h after 16 h. The 6-h lag period before increased tPA release was reproducible and independent of thrombin concentration. Thrombin inactivated with diisopropylfluorophosphate or hirudin did not induce an increase in tissue plasminogen activator levels. A 50-fold excess of diisopropylfluorophosphate-treated thrombin, which inhibits binding of active thrombin to endothelial cell high affinity binding sites, did not inhibit the thrombin-induced increase. It is concluded that proteolitically active thrombin causes an increase in the rate of release of tissue plasminogen activator from cultured human endothelial cells. The 6-h interval between thrombin treatment and enhanced tissue plasminogen activator release may reflect a delaying mechanism that transiently protects hemostatic plugs from the sudden increase in the local concentration of this fibrinolytic enzyme.

Neutrophil kinetics in man.

A method has been developed for measuring neutrophil cellularity in normal human bone marrow, in which the neutrophil-erythroid ratio was determined from marrow sections and marrow normoblasts were estimated by the erythron iron turnover. Neutrophil maturational categories, defined by morphologic criteria, were supported by autoradiographs of marrow flashed-labeled with 3H-thymidine. Correction for multiple counting error was empirically derived by counting serial sections through cells of each maturational category. The normal neutrophil-erythroid ratio in 13 normal human subjects was 1.5 +/- 0.07. The mean number of normoblasts in the same subjects was estimated to be 5.07 +/- 0.84 X 10(9) cells/kg. Total marrow neutrophils (X 10(9) cells/kg) were 7.70 +/- 1.20, the postmitotic pool (metamyelocytes, bands, and segmented forms) was 5.59 +/- 0.90 and the mitotic pool (promyelocytes + myelocytes) was 2.11 +/- 0.36. Marrow neutrophil ("total") production has been determined from the number of neutrophils comprising the postmitotic marrow pool divided by their transit time Transit time was derived from the appearance in circulating neutrophils of injected 3H-thymidine. The postmitotic pool comprised 5.59 +/- 0.90 X 10(9) neutrophils/kg, and the transit time was 6.60 +/- 0.03 days. From these data marrow neutrophil production was calculated to be 0.85 X 10(9) cells/kg per day. Effective production, measured as the turnover of circulating neutrophils labeled with 3H-thymidine, was 0.87 +/- 0.13 X 10(9) cells/kg per day. This value correlated well with the calculation of marrow neutrophil production. A larger turnover of 1.62 +/- 0.46 X 10(9) cells/kg per day was obtained when diisopropylfluorophosphate-32P was used to label circulating neutrophils. Studies using isologous cells doubly labeled with 3H-thymidine and diisopropylfluorophosphate-32P demonstrated a lower recovery and shorter t1/2 of the 32P label.

Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates.

The effects on thrombosis and hemostasis of thrombin-induced activation of endogenous protein C (PC) were evaluated in baboons. Thrombosis was induced by placing into arteriovenous shunts a segment of Dacron vascular graft, which generated arterial platelet-rich thrombus, followed by an expansion region of low-shear blood flow, which in turn accumulated fibrin-rich venous-type thrombus. Thrombosis was quantified by 111In-platelet imaging and 125I-fibrinogen accumulation. Intravenous infusion of alpha-thrombin, 1-2 U/kg-min for 1 h, increased baseline activated PC levels (approximately 5 ng/ml) to 250-500 ng/ml (P < 0.01). The lower thrombin dose, which did not deplete circulating platelets, fibrinogen, or PC, reduced arterial graft platelet deposition by 48% (P < 0.05), and platelet and fibrin incorporation into venous-type thrombus by > 85% (P < 0.01). Thrombin infusion prolonged the activated partial thromboplastin clotting time, elevated fibrinopeptide A (FPA), thrombin-antithrombin III complex (T:AT III), and fibrin D-dimer plasma levels (P < 0.01), but did not affect bleeding times. Thrombin's antithrombotic effects were blocked by infusing a monoclonal antibody (HPC-4) which prevented PC activation in vivo, caused shunt occlusion, increased the consumption of platelets and fibrinogen, elevated plasma FPA and T:AT III levels, and reduced factor VIII (but not factor V) procoagulant activity (P < 0.05). We conclude that activated PC is a physiologic inhibitor of thrombosis, and that activation of endogenous PC may represent a novel and effective antithrombotic strategy.

Marrow erythroid and neutrophil cellularity in the dog.

This paper describes a method for determining the number of marrow erythroid and neutrophil cells in which the cellularity of marrow sections was related to that of the total marrow by radioiron dilution. Tissue sections were prepared from methacrylate-embedded dog marrow biopsies, and neutrophils were identified by staining of their primary granules. After correction of direct section counts for multiple counting error, accurate neutrophil-erythroid ratios were established with a coefficient of variation of less than 10 percent when 10-4 cells were examined. An average neutrophil-erythroid ratio of 1.2 was found in six normal dogs. The total number of nucleated red cells in the dog was 5.48 plus or minus 0.78 times 10-9/kg (plus or minus 1 SD), and the corresponding erythron iron turnover was 0.90 plus or minus 0.11 mg Fe/100 ml whole blood/day. The total number of marrow neutrophils, derived from the neutrophil-erythroid ratio, was 6.6 plus or minus 0.59 times 10-9 cells/kg, of which 1.4 were promyelocytes and myelocytes, 2.3 were metamyelocytes and bands, and 3.0 were segmented neutrophils. Leukopheresis studies were carried out in six dogs to confirm the accuracy of these cellular measurements. Marrow counts showed a mean decrease of 22.7 times 10-9 cells or 35 percent of the postmitotic neutrophil pool, and it was calculated that 10.2 times 10-9 additional cells had been taken from already circulating blood. This estimated deficit of 32.9 times 10-9 was almost identical to the 33 times 10-9 cells actually counted in the removed blood.

Glutathione redox cycle protects cultured endothelial cells against lysis by extracellularly generated hydrogen peroxide.

We have examined the role of the glutathione redox cycle as an antioxidant defense mechanism in cultured bovine and human endothelial cells by disrupting the glutathione redox cycle at several points. Endothelial glutathione reductase was selectively inhibited with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU). Cellular stores of reduced glutathione were depleted by reaction with diethylmaleate (DEM) or 1-chloro-2,4-dinitrobenzene (CDNB) or by inhibition of glutathione synthesis with buthionine sulfoximine (BSO). Whereas several strains of untreated bovine and human endothelial cells were resistant to lysis by enzymatically generated hydrogen peroxide, BCNU-treated cells were readily lysed in a time- and dose-dependent manner. Glucose-glucose oxidase-mediated lysis of BCNU-treated bovine endothelial cells was catalase-inhibitable and directly related to BCNU concentration and endogenous glutathione reductase activity. Pretreatment of bovine endothelial cells with BCNU did not potentiate lysis by distilled water, calcium ionophore, lipopolysaccharide, or hypochlorous acid. Depletion of cellular reduced glutathione by reaction with DEM or CDNB or by inhibition of glutathione synthesis by BSO also potentiated endothelial lysis by enzymatically generated hydrogen peroxide. Inhibition of endothelial glutathione reductase by BCNU or depletion of reduced glutathione by BSO increased endothelial susceptibility to lysis by hydrogen peroxide generated by phorbol myristate acetate-activated neutrophils. We conclude that the glutathione redox cycle plays an important role as an endogenous antioxidant defense mechanism in cultured endothelial cells.

Neutrophil-mediated endothelial injury in vitro mechanisms of cell detachment.

Neutrophil-mediated endothelial injury was assessed in vitro using assays of cell lysis and cell detachment. Activation of human peripheral blood neutrophils adherent to human umbilical vein endothelial cell monolayers by serum-treated zymosan produced dose-dependent endothelial cell detachment without concomitant cell lysis. This injury was inhibited by neutral protease inhibitors, but not by catalase or superoxide dismutase. Neutrophils from a patient with chronic granulomatous disease also produced endothelial cell detachment when activated by serum-treated zymosan similar to normal neutrophils. Endothelial detachment was also produced by cell-free postsecretory media from activated neutrophils or by partially purified human neutrophil granule fraction and was inhibitable by tryptic, elastase, and serine protease inhibitors, but not by an acid protease inhibitor. Analysis of iodinated endothelial cell surface proteins that had been exposed to partially purified neutrophil granule fraction showed complete loss of proteins migrating in the region of fibronectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This result was prevented in the presence of neutral protease inhibitors. We conclude that neutrophil-derived neutral proteases mediate endothelial cell detachment in vitro through digestion of endothelial cell surface proteins including fibronectin.

Effects of monoclonal antibodies against the platelet glycoprotein IIb/IIIa complex on thrombosis and hemostasis in the baboon.

To assess the hemostatic consequences and antithrombotic effectiveness of blocking the platelet glycoprotein (GP) IIb/IIIa receptor for fibrinogen and other adhesive glycoproteins in vivo, well characterized murine monoclonal antibodies against the platelet GP IIb/IIIa complex, AP-2 and LJ-CP8, were infused intravenously into baboons. Four animals each received doses of 0.2, 0.4, and 1.0 mg/kg of purified AP-2 IgG, and three animals were given 1.0 mg/kg of the F(ab)2 fragment of AP-2. Five additional animals were given 10 mg/kg LJ-CP8 IgG. At the highest dose, radiolabeled AP-2 IgG bound to an average of 33,000 sites on the circulating platelets. Serial measurements included platelet count, bleeding time, platelet aggregation (induced by ADP, collagen, and gamma-thrombin), and 111In-platelet deposition onto Dacron vascular grafts. Bleeding times were markedly prolonged after injection of 1.0 mg/kg AP-2 IgG (19.2 +/- 3.4 min), 1.0 mg/kg AP-2 F(ab)2 (16.5 +/- 1.8 min), and 10 mg/kg LJ-CP8 (greater than 30 min) vs. control studies (4.6 +/- 0.2 min), and remained prolonged for 48 h. With each antibody platelet aggregation was initially reduced or absent, with partial recovery over 48 h in a manner that was inversely related to dose. AP-2, both whole IgG and F(ab)2 fragment, but not LJ-CP8, caused a dose-dependent reduction (20-46%) in the circulating platelet count over 24 h. Neither AP-2 nor LJ-CP8 caused a reduction in intraplatelet platelet factor 4, beta-thromboglobulin, or [14C]serotonin. Graft-associated platelet thrombus formation was reduced by 73% (1.0 mg/kg AP-2 IgG and 10 mg/kg LJ-CP8) and 53% (1.0 mg/kg AP-2 F(ab)2) relative to control values. In contrast, neither heparin (100 U/kg) nor aspirin (32.5 mg/kg twice a day) showed antithrombotic efficacy in this model. Thus, antibodies that functionally alter the platelet GP IIb/IIIa complex may produce immediate, potent, and transient, antihemostatic, and antithrombotic effects.

Variations in circulating catecholamines fail to alter human platelet alpha-2-adrenergic receptor number or affinity for [3H]yohimbine or [3H]dihydroergocryptine.

A series of studies were performed to determine the relationship between physiologic levels of circulating plasma norepinephrine and epinephrine and human platelet alpha-2 binding site number and the affinity (KD) of these sites for antagonist radioligands. In one study, alpha-2-adrenergic binding site number and affinity were compared using both [3H]yohimbine and [3H]dihydroergocryptine as radioligands. There was good absolute and relative comparison for binding site number, but only a relative relationship for KD. In 46 normal subjects, there was no significant relationship between site number or KD and age, plasma epinephrine, or plasma norepinephrine concentration. Even after plasma epinephrine was raised nearly 20-fold by means of an intravenous infusion for 4 h in seven normal subjects, neither sites (608 +/- 68 vs. 567 +/- 120 sites/platelet) nor KD (2.01 +/- 0.94 vs. 2.14 +/- 1.15 nM) were significantly changed. Similarly, neither sites (445 +/- 55 vs. 421 +/- 53 sites/platelet) nor KD (1.44 +/- 0.29 vs. 2.10 +/- 0.75 nM) were significantly changed in six normal subjects when plasma norepinephrine levels increased during oral administration of prazosin for 1 wk. Thus, in a cross-sectional analysis and after a change in plasma catecholamine concentrations, there was no relationship in normal subjects between platelet alpha-2 binding site number or affinity of these sites for antagonist radioligands and the circulating catecholamine levels to which the platelets were exposed. In a group (n = 7) of patients who lack epinephrine-induced platelet aggregation due to abnormal thrombopoiesis, binding site number was decreased (304 +/- 36 vs. 572 +/- 29 sites/platelet, P less than 0.001) and KD tended to be greater (8.69 +/- 2.44 vs. 5.40 +/- 0.31 nM, P = NS) than in normal subjects (n = 46), despite having similar plasma catecholamine levels. There was no difference in binding site number (491 +/- 116 sites/platelet) and KD (5.61 +/- 0.84 nM) in patients (n = 5) with autonomic insufficiency and low levels of upright plasma norepinephrine when compared with the normal subjects. Two patients were examined before and after the removal of a pheochromocytoma. Their binding site number and KD were normal before the operation and essentially unchanged after the tumor removal and fall of plasma catecholamines. Thus, this study demonstrates that within the physiologic and pathophysiologic range of plasma catecholamines (in men), there is no relationship between the circulating catecholamine concentration and either platelet alpha-2 adrenergic binding site number or the affinity of these sites for antagonist radioligands.

Biochemical studies of two patients with the gray platelet syndrome. Selective deficiency of platelet alpha granules.

The biochemistry of platelets from two unrelated patients with the gray platelet syndrome, a deficiency of platelet alpha-granules, has been evaluated. Ultrastructural studies of their platelets revealed the number of alpha-granules to be less than 15% of normal, whereas the number of dense bodies was within normal limits. Platelets from both patients had severe deficiencies of platelet factor 4 and beta-thromboglobulin (less than 10% of normal). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a marked deficiency of thrombin-sensitive protein in both patients. Analysis of the platelet-derived growth factor in one patient showed it was also markedly reduced. Levels of lysosomal enzymes, adenine nucleotides, serotonin, and catalase, and conversion of arachidonic acid by the lipoxygenase and cyclo-oxygenase enzymes, were within normal limits. The results provide important evidence to define the contents of alpha-granules and to differentiate these contents from the contents of lysosomal granules, dense bodies, and peroxisomes. Functional studies of these platelets showed deficiencies in ADP, thrombin, and collagen aggregation. The results suggest that alpha-granules or their contents make a contribution to normal platelet aggregation.

Reduction in vascular lesion formation by hirudin secreted from retrovirus-transduced confluent endothelial cells on vascular grafts in baboons.

BACKGROUND: The hypothesis that thrombin mediates the formation of neointimal vascular lesions at sites of mechanical vascular injury has been tested in baboons by measurement of the effects of hirudin delivered by retrovirus-transduced hirudin-secreting vascular endothelial cells (ECs) lining surgically implanted arterial vascular grafts (AVGs). METHODS AND RESULTS: The antithrombotic efficacy of baboon ECs transduced with cDNA encoding hirudin was assessed in vitro and in vivo on thrombogenic segments in chronically exteriorized femoral arteriovenous (AV) shunts. Bilateral brachial AVGs lined with hirudin-transduced versus nonhirudin control ECs at confluent density were surgically implanted, and vascular lesion formations at distal graft-vessel anastomoses were compared after 30 days. Hirudin-transduced ECs secreted 20+/-6 ng x 10(6) cells(-1) x 24 h(-1) (range, 14 to 24 ng x 10(6) cells(-1) x 24 h(-1)) hirudin in supernatants of static cultures. Hirudin-secreting ECs on segments of collagen-coated graft interposed in chronic AV shunts decreased the accumulation of (111)In-labeled platelets to 0.52+/-0.34 x 10(9) platelets, compared with 0.82+/-0.49 x 10(9) platelets in controls (P = 0.03) and reduced platelet deposition in propagated thrombotic tails extending downstream from segments of vascular graft from 1.38+/-0.41 x 10(9) platelets in controls to 0.59+/-0.22 x 10(9) platelets (P = 0.04). ECs recovered from 30-day AVG implants generated 17+/-9 ng x 10(6) cells(-1) x 24 h(-1) (range, 9 to 25 ng x 10(6) cells(-1) x 24 h(-1)) hirudin. Hirudin-secreting ECs reduced neointimal lesion formation at distal graft-vessel anastomoses, ie, 1.02 mm(2) (range, 0.88 to 1.95 mm(2)) versus 1.82 mm(2) (range, 0.88 to 2.56 mm(2)) in contralateral AVGs bearing nonhirudin control ECs (P<0.01). CONCLUSIONS: Viral vector-directed secretion of hirudin from ECs lining implanted AVGs significantly reduces the formation of thrombus and neointimal vascular lesions.

Pharmacology of platelet inhibitors.

Although many drugs have inhibitory effects on platelet function, none of them inhibits all of the mechanisms that may be involved in the various forms of thrombosis. Choice of suitable drugs is hampered by lack of full knowledge concerning the reactions that make the major contributions to the formation of arterial thrombi at sites of repeated vessel wall injury or on atherosclerotic lesions. Drugs such as aspirin that inhibit the arachidonate pathway in platelets can only be expected to be effective against thromboembolic events in which the generation of thromboxane A2 plays a major part. If thrombin and fibrin formation are dominant, oral anticoagulant agents or heparin should be beneficial; thus, experimental evidence indicates that with repeated vessel wall injury, the formation of platelet fibrin thrombi on the vessel wall is probably influenced more by inhibitors of thrombin generation than by the subendothelial constituents such as collagen. Agents like prostacyclin that raise platelet cyclic adenosine monophosphate (AMP) levels in platelets by stimulating adenylate cyclase are potent inhibitors of the reaction of platelets to all aggregating and release-inducing stimuli, but these agents are not suitable for long-term administration. The effect of dipyridamole on platelet cyclic AMP levels is weak, and this drug may act through other effects on platelets or on other cells. Indeed, several of the drugs that have been tested in clinical trials may exert their effects through unrecognized mechanisms. Many combinations of drugs have been used to affect platelets or platelets and coagulation. This practice has been based on the theory that because several mechanisms may be involved in thrombus formation, combinations of drugs that inhibit different mechanisms may be beneficial.

Effects of angiotensin converting enzyme inhibition with cilazapril on intimal hyperplasia in injured arteries and vascular grafts in the baboon.

To determine the importance of angiotensin converting enzyme (ACE) activity in the development of arterial proliferative lesions in a primate model, the response to vascular injury was studied in five baboons treated with oral cilazapril (20 mg/kg/day) and in five untreated control animals. Each animal underwent three procedures: 1) carotid artery endarterectomy, 2) balloon catheter deendothelialization of the superficial femoral artery, and 3) surgical placement of bilateral aorto-iliac expanded polytetrafluoroethylene (Gore-Tex) vascular grafts. Cilazapril therapy was initiated 1 week preoperatively and continued throughout the study interval. At 1 and 3 weeks postoperatively, plasma ACE activity was inhibited by more than 96% versus control values. After animals were killed at 3 months, injured vessel and graft segments were evaluated morphometrically. Although the response between animals was variable, average cross-sectional areas of neointima did not differ between the cilazapril-treated and control groups at sites of carotid endarterectomy (0.26 +/- 0.12 versus 0.34 +/- 0.17 mm2, respectively; p greater than 0.5), femoral artery ballooning (0.15 +/- 0.08 versus 0.11 +/- 0.01 mm2; p greater than 0.5), or at graft anastomoses (1.86 +/- 0.50 versus 1.72 +/- 0.50 mm2; p greater than 0.5). Thus, cilazapril did not reduce intimal thickening over 3 months in these primate arterial injury models. However, a possible beneficial effect of cilazapril, which might be apparent at earlier time points or with larger animal groups, cannot be excluded.

Platelets in thrombotic disorders: quantitative and qualitative platelet disorders predisposing to arterial thrombosis.

Endogenous thrombopoietin (TPO) stimulates platelet production in nonhuman primates by inducing dose-dependent megakaryocyte development from early marrow hematopoietic progenitors and subsequent proliferation and endoreduplication. In nonhuman primates, recombinant human TPO, nonpegylated or pegylated rHu megakaryocyte growth and development factor produce log-linear responses in peak peripheral platelet counts (or peripheral platelet mass turnover) and marrow megakaryocyte volume, ploidy, number, and mass. Mpl ligands can support normal peripheral platelet concentrations after myelosuppressive chemotherapy in baboons, and correct the thrombocytopenia in human immunodeficiency virus-infected chimpanzees. Whereas mpl ligands do not induce platelet aggregation in vitro, mpl ligands enhance aggregatory responsiveness of platelets to physiologic agonists both in vitro and transiently ex vivo after treatment with mpl ligands in nonhuman primates. However, platelet recruitment into forming thrombus is not augmented by these agents when evaluated in quantitative rabbit or baboon models of platelet-dependent thrombus formation, except for the effect of platelet concentration, per se. These findings indicate that appropriate dosing of mpl ligands prevents thrombocytopenia without increasing the risk of platelet-dependent thrombo-occlusive complications.