Long-term outcome after spinal cord infarctions.

OBJECTIVES: To investigate long-term outcome in patients with spontaneous spinal cord infarctions and secondly to compare outcome with that of patients with cerebral infarction. MATERIAL AND METHODS: The study includes 30 patients with spinal cord infarction discharged between 1995 and 2010. Surviving patients were contacted by telephone and sent a questionnaire. Data on employment, function, depression, fatigue, pain, and quality of life were obtained and compared to similar data obtained from a group of patients with cerebral infarction. RESULTS: Seven patients with spinal cord infarction had died after a mean follow-up of 7.1 years. Mortality was associated with poor functioning in the acute phase. Thirteen of 20 responding patients were able to walk. Compared to patients with cerebral infarction, patients with spinal cord infarction had significantly lower mortality, poorer functioning, higher re-employment rate, and more pain. CONCLUSION: Many patients with spinal cord infarction experience significant improvement. Even though functional outcome is worse, the mortality rate is lower and the frequency of re-employment higher among patients with spinal cord infarction compared to patients with cerebral infarction.

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.

RGDV peptide selectively inhibits platelet-dependent thrombus formation in vivo. Studies using a baboon model.

Since platelet hemostatic functions are mediated in part through the binding of adhesive proteins containing an RGD (Arg-Gly-Asp) recognition sequence, and since platelet reactions may be inhibited in vitro by RGD-containing peptides, we assessed in vivo the antithrombotic activity of RGDV (Arg-Gly-Asp-Val) tetrapeptide using a baboon thrombosis model. Thrombus formation was induced by a device consisting of a tubular segment coated with type I collagen, followed by two regions of expanded diameter exhibiting disturbed flow and stasis. The thrombogenic device was incorporated into femoral arteriovenous shunts under conditions of intermediate wall shear rate (100 s-1). Thrombus formation was measured by scintillation camera imaging of 111In-platelets and by counting of 125I-fibrinogen/fibrin. Thrombus that formed on the collagen substrate was rich in platelets, while thrombus formed in the disturbed flow regions was rich in fibrin and red cells. RGDV peptide was infused proximal to the thrombogenic device to maintain local plasma concentrations of 25, 50, and 100 microM. Infused RGDV decreased the accumulation of both platelets and fibrin on the collagen substrate in a dose-response manner. At the highest dose platelet and fibrin deposition after 40 min was reduced by greater than 80% (P less than 0.01). In the region of disturbed flow, RGDV (100 microM) reduced platelet deposition by 85% (P less than 0.01) but did not reduce the accumulation of fibrin (P less than 0.3). Similarly, the peptide inhibited the release of granular proteins from platelets associated with thrombus (platelet factor 4, beta-thromboglobulin; P less than 0.01), but did not prevent the appearance of fibrinopeptide A in circulating blood (P greater than 0.1). No systemic alterations in blood pressure, bleeding time, or platelet aggregation ex vivo were produced by locally infused RGDV. The antithrombotic effects of RGDV peptide disappeared within 5 min after discontinuing the infusion. In control studies infused RGEV (Arg-Gly-Glu-Val, 100 microM) showed no antithrombotic activity. Thus, RGDV selectively blocks platelet-dependent thrombus formation in vivo.

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.

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.

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.

Limited generation of activated protein C during infusion of the protein C activator thrombin analog W215A/E217A in primates.

Anticoagulation with activated protein C (APC) reduces the mortality of severe sepsis. We investigated whether the circulating protein C (PC) pool could be utilized for sustained anticoagulation by endogenous APC. To generate APC without procoagulant effects, we administered the anticoagulant thrombin mutant W215A;E217A (WE) to baboons. In preliminary studies, administration of high dose WE (110 microg kg(-1) i.v. bolus every 120 min; n = 2) depleted PC levels by 50% and elicited transient APC bursts and anticoagulation. The response to WE became smaller with each successive injection. Low dose WE infusion (5 microg kg(-1) loading + 5 microg kg(-1) h(-1) infusion; n = 5) decreased plasma PC activity by 15%, from 105% to 90%, to a new equilibrium within 60 min. APC levels increased from 7.5 ng mL(-1) to 86 ng mL(-1) by 40 min, then declined, but remained elevated at 34 ng mL(-1) at 240 min. A 22-fold higher dose WE (n = 5) decreased PC levels to 60% by 60 min without significant further depletion in 5 h. The APC level was 201 ng mL(-1) at 40 min and decreased to 20 ng mL(-1) within 120 min despite continued activator infusion. Co-infusion of WE and equimolar soluble thrombomodulin (n = 5) rapidly consumed about 80% of the PC pool with significant temporal increase in APC generation. In conclusion, low-grade PC activation by WE produced sustained, clinically relevant levels of circulating APC. Limited PC consumption in WE excess was consistent with the rapid depletion of cofactor activity before depletion of the PC zymogen. Reduced utilization of circulating PC might have similar mechanism in some patients.

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.

Local delivery of a synthetic antithrombin with a hydrogel-coated angioplasty balloon catheter inhibits platelet-dependent thrombosis.

OBJECTIVES: This study evaluated the efficacy of local administration of an antithrombin agent with a hydrogel-coated percutaneous transluminal coronary angioplasty balloon catheter. BACKGROUND: Intravenous infusion of antithrombin compounds has been shown to inhibit platelet-dependent thrombosis. However, hemorrhage is a common side effect associated with the systemic administration of antithrombin compounds. METHODS: The potent, irreversible thrombin inhibitor D-Phe-L-Pro-L-Arginyl chloromethyl ketone (PPACK) was used to inhibit thrombus formation in chronic porcine arteriovenous shunts. Platelet deposition was quantitated with gamma camera imaging of 111In-labeled platelets. RESULTS: Intravenous administration of PPACK in swine, in doses sufficient to maximally inhibit thrombus formation, was associated with prolongation of bleeding parameters. The inhibition of thrombosis associated with intravenous PPACK was dose related. The amount of intravenous PPACK necessary for maximal inhibition of thrombus formation for a period of 45 min was 16.9 mg. In contrast, local delivery of PPACK with a hydrogel-coated angioplasty balloon deployed at the site of the thrombus inhibited platelet deposition for at least 45 min after the balloon was removed. Using 3H-labeled PPACK, the calculated amount of PPACK delivered was 33.5 micrograms. There was no change in bleeding time or activated partial thromboplastin time when swine received an intravenous bolus greater than the total amount of PPACK adsorbed onto the balloon (70 micrograms). CONCLUSIONS: These results suggest that in this model, a hydrogel-coated coronary angioplasty balloon catheter can be used to deliver enough antithrombin agent to inhibit platelet-dependent thrombosis for at least 45 min at doses that are several orders of magnitude less than those required for systemic administration. In addition, local delivery can provide effective inhibition of thrombus formation without alteration of bleeding parameters.

Platelet reactivity in depressed patients treated with paroxetine: preliminary findings.

BACKGROUND: Alterations in platelet reactivity have been previously posited to underlie the increased vulnerability of patients with depression to ischemic heart disease (IHD). The present study sought to determine whether the increased platelet reactivity associated with major depression is reduced after antidepressant treatment. METHODS: Patients diagnosed as having DSM-IV major depression (n = 15) (mean age, 37 +/- 7 years; range, 23-48 years) and 12 normal comparison subjects (mean age, 36 +/- 7; range, 23-48 years) were recruited. None of the controls or depressed group had evidence of IHD; 10 of 15 patients who were depressed had 1 or more traditional IHD risk factors. In vivo platelet activation, secretion, and dose-response aggregation of the controls and patients was measured after overnight bedrest under basal conditions, and after a mild exercise challenge. After 6 weeks of open-label treatment with the selective serotonin reuptake inhibitor paroxetine (20 mg/d), the patients with depression were readmitted and procedures of the first General Clinical Research Center admission repeated. RESULTS: In comparison with the control group, the depressed group exhibited greater procoagulant activity as detected by increased platelet binding of the monoclonal antibodies anti-ligand-induced binding site and GA6, and increased plasma concentrations of platelet factor 4 under basal conditions. After paroxetine treatment, the patients with depression exhibited significant reductions in all 3 parameters. CONCLUSIONS: Normalization of platelet activation is associated with paroxetine treatment of patients with depression. Because this study design did not allow for the determination of whether this effect of paroxetine on platelet function is caused by a direct effect of the drug or placebo or, alternatively, because of recovery from depression, studies containing a placebo and/or psychotherapy treatment arm may resolve this issue.

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.

Alternative splicing of the mRNA encoding baboon glycoprotein receptor GPIIb.

The cloning and characterization of cDNAs encoding the cell-surface-specific integrin receptors of baboon platelets has been undertaken to provide species-specific probes. These will be used to investigate the expression and distribution of these receptors among primate species. Clones GPIIb-16 and GPIIb-3, encoding portions of the baboon glycoprotein GPIIb, were isolated from a cDNA library derived from baboon platelet mRNA. GPIIb-3 includes an insert of 43 bp, when compared to GPIIb-16 or human GPIIb. This insert is the result of alternative processing of mRNA. The probable origin of the inserted bases is the 3' end of the intron preceeding exon 28 of the gene. A different product of alternative splicing has been reported in this same region of the human GPIIb sequence, suggesting that this location is susceptible to wobble in the intron-exon junctions. The projected shift in the reading frame of the baboon GPIIb-3 cDNA would give a radically altered C terminus of the deduced amino-acid sequence, and the possibility of a novel functional peptide on the platelet surface.

Human and baboon integrin beta 5 subunit-encoding mRNAs have alternative polyadenylation sites.

A cDNA probe, encoding part of the human integrin polypeptide, GPIIIa (beta 3), was used to screen a cDNA library derived from baboon smooth muscle polyadenylated mRNA. One cross-hybridizing clone, lacking the baboon equivalent of 478 bp of the human sequence, was found to be 98% similar to a human cDNA encoding the beta 5-chain of the receptor for vitronectin, a cell adhesion molecule. The baboon mRNA terminates at 3' position 212 nucleotides upstream from the polyadenylation site of the aligned human mRNA.

Characterization of a cDNA encoding the beta-chain of baboon receptor glycoprotein GPIb.

A cDNA probe, encoding part of the baboon integrin polypeptide, GPIIb, was used to screen a cDNA library derived from baboon platelet polyadenylated mRNA. One cross-hybridizing clone was found to be 94% similar to a human cDNA encoding the beta-chain of the receptor for von Willebrand factor, a major component of the platelet-subendothelium attachment mechanism.

Potential new targets for antithrombotic therapy.

Thrombosis is the collective term for diseases caused by the localized accumulation of circulating blood elements within the vasculature that result in vessel occlusion. Conventional antithrombotic drugs can inhibit thrombus growth by targeting coagulation pathways (e.g., heparin, warfarin) or platelet-dependent mechanisms (e.g., aspirin, clopidogrel). Thrombolytic agents (e.g., streptokinase) are used to degrade thrombi in situ, thereby restoring the blood flow. Despite advances, the search for new strategies continues because existing treatments impair hemostasis, and must be administered at dose levels that do not achieve maximum efficacy. Only a few drugs are used at markedly efficacious doses, for short periods of time in closely watched clinical situations, such as interventional cardiology and surgery. Ideally, new targets for therapy would lead to the development of agents that are specific for thrombus-forming mechanisms without affecting hemostasis. In the absence of such agents, new products should preferentially inhibit the thrombotic process at doses that are relatively safe. The symptomatology of naturally occurring or experimentally-induced alterations of relevant hemostatic pathways can serve as basis for target selection. Hemostatic disorders that are compatible with life, do not pose a significantly increased risk of bleeding, but potentially protect against thrombosis provide guidance for rational design strategies. Theoretical considerations and recent experimental data suggest that: 1) inhibition of intrinsic coagulation pathway activity, 2) reduction of circulating platelet count, or 3) activation or enhancement of endogenous protein C or thrombolytic pathways could improve antithrombotic therapy.

Localizing antithrombotic and vasodilatory activity with a novel, ultrafast nitric oxide donor.

Reaction of nitric oxide (NO) with L-proline in methanolic sodium methoxide yields a diazeniumdiolate product, C5H7N3O4Na2.CH3OH (PROLI/NO), that can be stabilized in basic solution but that dissociates to proline (1 mol) and NO (2 mol) with a half-life of only 1.8 s at pH 7.4 and 37 degrees C. This kinetic behavior has allowed the generation of highly localized antiplatelet and vasodilatory effects. By infusing solutions containing 4 microM PROLI/NO in 0.1 M sodium hydroxide at the rate of 1 nmol.min-1 immediately upstream from a polyester vascular graft in the unheparinized baboon circulatory system, for example, platelet deposition at the normally thrombogenic graft surface was substantially reduced relative to controls receiving only 0.1 M sodium hydroxide. In a second study, infusion of PROLI/NO into the right atrium of sheep with induced pulmonary hypertension selectively dilalated the lung vasculature, dose-dependently reducing the pulmonary arterial pressure by as much as 9 mmHg with no observable effect on the systemic arterial pressure at an infusion rate of up to 24 nmol.kg-1.min-1. PROLI/NO could also be formulated as an insoluble polymer blend that released NO smoothly for prolonged periods. The results suggest that localized delivery of diazeniumdiolates such as PROLI/NO which generate NO with extreme rapidity on entering the blood stream may hold considerable promise for inhibition of thrombus formation, selective dilation of the vasculature, and other research and clinical applications.

Synthesis and biological evaluation of myoseverin derivatives: microtubule assembly inhibitors.

Myoseverin, a trisubstituted purine, inhibits microtubule assembly in vitro, interferes with normal mitotic spindle assembly, and arrests the cell cycle in mitosis in U937 cells. We synthesized a variety of myoseverin derivatives and screened them for inhibition of spindle assembly in Xenopus egg extracts and for microtubule disassembly in vitro. Selected compounds were tested against 60 cancer cell lines at the National Cancer Institute as possible anticancer drug candidates.

Nitric oxide-releasing polymers containing the [N(O)NO]- group.

Ions of structure X[N(O)NO]- display broad-spectrum pharmacological activity that correlates with the rate and extent of their spontaneous, first-order decomposition to nitric oxide when dissolved. We report incorporation of this functional group into polymeric matrices that can be used for altering the time course of nitric oxide release and/or targeting it to tissues with which the polymers are in physical contact. Structural types prepared include those in which the [N(O)NO]- group is attached to heteroatoms in low molecular weight species that are noncovalently distributed throughout the polymeric matrix, in groupings pendant to the polymer backbone, and in the polymer backbone itself. They range in physical form from films that can be coated onto other surfaces to microspheres, gels, powders, and moldable resins. Chemiluminescence measurements confirm that polymers to which the [N(O)NO]- group is attached can serve as localized sources of nitric oxide, with one prototype providing sustained NO release for 5 weeks in pH 7.4 buffer at 37 degrees C. The latter composition, a cross-linked poly-(ethylenimine) that had been exposed to NO, inhibited the in vitro proliferation of rat aorta smooth muscle cells when added as a powder to the culture medium and showed potent antiplatelet activity when coated on a normally thrombogenic vascular graft situated in an arteriovenous shunt in a baboon's circulatory system. The results suggest that polymers containing the [N(O)NO]- functional group may hold considerable promise for a variety of biomedical applications in which local delivery of NO is desired.

Baboon models of acute arterial thrombosis.

In conclusion, we believe that the baboon offers many advantages as an experimental animal to study vascular disease, thrombus formation and dissolution, the effects of mediating variables, and the relative efficacy of therapeutic interventions. Each specific application for testing therapeutic agents may require testing in different model systems. For example, the AV vascular graft model is efficient, cost effective and well adapted to study interventions for acute arterial thrombosis. As the antithrombotic products of genetic engineering and molecular biology emerge, it will be increasingly important to have relevant, reproducible, and quantitative approaches to evaluate their effects in vivo.

Studies of suloctidil in experimental thrombosis in baboons.

Suloctidil has been evaluated in the baboon for its antithrombotic efficacy using models of both acute and chronic arterial thrombogenesis. Acute thrombus formation was initiated by Dacron vascular grafts inserted as extension segments into chronic arteriovenous shunts. 111In-platelet deposition was measured by scintillation camera imaging for one hour. The results after oral administration of suloctidil (100 mg/kg/d in two divided doses) were not different from control studies. Moreover, concurrent heparin anticoagulation did not affect 111In-platelet deposition compared with control data. In contrast, ticlopidine (20 mg/kg/d) significantly decreased platelet deposition that was reduced further by the addition of heparin. Chronic arterial-thromboembolism was initiated by segments of polyurethane (Biomer) cannula introduced into chronic arteriovenous shunts. Thrombus formation by the polyurethane cannula was measured as 111In-platelet turnover (corrected for removal of senescent platelets). Cannula platelet consumption was unaffected by suloctidil (20 mg/kg/d given in two divided doses for two days preceding and throughout the period of platelet survival measurement). In contrast, dipyridamole (10 mg/kg/d) and sulfinpyrazone (100 mg/kg/d) completely interrupted cannula platelet consumption. We conclude that suloctidil probably has little or no effect on platelet-dependent thrombus formation.