Walter J. Janse van Rensburg

Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon ( Papio ursinus )

Thrombotic thrombocytopenic purpura (TTP) is the prototypical microangiopathy characterized by disseminated microthromboses, hemolytic anemia, and ultimately organ dysfunction. A link with deficiency of the von Willebrand factor-cleaving protease (ADAMTS13) has been demonstrated, but additional genetic and/or environmental triggers are thought to be required to incite acute illness. Here we report that 4 days of ADAMTS13 functional inhibition is sufficient to induce TTP in the baboon (Papio ursinus), in the absence of inciting triggers because injections with an inhibitory monoclonal antibody (mAb) consistently (n = 6) induced severe thrombocytopenia (< 12 × 10(9)/L), microangiopathic hemolytic anemia, and a rapid rise in serum lactate dehydrogenase. Immunohistochemical staining revealed the characteristic disseminated platelet- and von Willebrand factor-rich thrombi in kidney, heart, brain, and spleen but not lungs. Prolonged inhibition (14 days, n = 1) caused myocardial ischemic damage and asplenia but not death. Control animals (n = 5) receiving equal doses of a noninhibitory anti-ADAMTS13 mAb remained unaffected. Our results provide evidence for a direct link between TTP and ADAMTS13 inhibition and for a mild disease onset. Furthermore, we present a reliable animal model of this disease as an opportunity for the development and validation of novel treatment strategies.

Evaluation of efficacy and safety of the anti-vWF Nanobody ALX-0681 in a preclinical baboon model of acquired thrombotic thrombocytopenic purpura

ALX-0681 is a therapeutic Nanobody targeting the A1-domain of VWF. It inhibits the interaction between ultra-large VWF and platelet GpIb-IX-V, which plays a crucial role in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). In the present study, we report the efficacy and safety profile of ALX-0681 in a baboon model of acquired TTP. In this model, acute episodes of TTP are induced by administration of an ADAMTS13-inhibiting mAb. ALX-0681 completely prevented the rapid onset of severe thrombocytopenia and schistocytic hemolytic anemia. After induction of TTP, platelet counts also rapidly recovered on administration of ALX-0681. This effect was corroborated by the full neutralization of VWF activity. The schistocytic hemolytic anemia was also halted and partially reversed by ALX-0681 treatment. Brain CT scans and post mortem analysis did not reveal any sign of bleeding, suggesting that complete neutralization of VWF by ALX-0681 under conditions of thrombocytopenia was not linked with an excessive bleeding risk. The results obtained in this study demonstrate that ALX-0681 can successfully treat and prevent the most important hallmarks of acquired TTP without evidence of a severe bleeding risk. Therefore, ALX-0681 offers an attractive new therapeutic option for acquired TTP in the clinical setting.

Inhibition of von Willebrand factor-platelet glycoprotein Ib interaction prevents and reverses symptoms of acute acquired thrombotic thrombocytopenic purpura in baboons

The pathophysiology of thrombotic thrombocytopenic purpura (TTP) can be explained by the absence of active ADAMTS13, leading to ultra-large von Willebrand factor (UL-VWF) multimers spontaneously interacting with platelets. Preventing the formation of UL-VWF-platelet aggregates therefore is an attractive new treatment strategy. Here, we demonstrate that simultaneous administration of the inhibitory anti-VWF monoclonal antibody GBR600 and the inhibitory anti-ADAMTS13 antibody 3H9 to baboons (prevention group) precluded TTP onset as severe thrombocytopenia and hemolytic anemia were absent in these animals. In addition, partial VWF inhibition was not enough to prevent thrombocytopenia, demonstrating the specificity of this therapeutic strategy. GBR600 treatment of baboons during acute TTP (treatment group) resulted in a rapid recovery of severe thrombocytopenia similar to the platelet count increases observed in TTP patients treated by plasma exchange. Baboons in the control group only injected with 3H9 developed early stages of TTP as previously described. Hence, inhibiting VWF-GPIb interactions is an effective way to prevent and treat the early symptoms of acquired TTP in baboons.

Polyunsaturated fatty acids cause apoptosis in C. albicans and C. dubliniensis biofilms

BACKGROUND Polyunsaturated fatty acids (PUFAs) have antifungal properties, but the mode by which they induce their action is not always clear. The aim of the study was to investigate apoptosis as a mode of action of antifungal PUFAs (stearidonic acid, eicosapentaenoic acid and docosapentaenoic acid) which are inhibitory towards biofilm formation of C. albicans and C. dubliniensis. METHODS Candida biofilms were grown in the absence or presence of 1mM PUFAs (linoleic acid, stearidonic acid, eicosapentaenoic acid, docosapentaenoic acid) for 48h at 37°C. The effect of these PUFAs on the membrane fatty acid profile and unsaturation index, oxidative stress, mitochondrial transmembrane potential and apoptosis was evaluated. RESULTS When biofilms of C. albicans and C. dubliniensis were exposed to certain PUFAs there was an increase in unsaturation index of the cellular membranes and accumulation of intracellular reactive oxygen species (ROS). This resulted in apoptosis, evidenced by reduced mitochondrial membrane potential and nuclear condensation and fragmentation. The most effective PUFA was stearidonic acid. CONCLUSIONS The resultant cell death of both C. albicans and C. dubliniensis is due to apoptosis. GENERAL SIGNIFICANCE Due to the increase in drug resistance, alternative antifungal drugs are needed. A group of natural antifungal compounds is PUFAs. However, understanding their mechanisms of action is important for further use and development of these compounds as antifungal drugs. This paper provides insight into a possible mode of action of antifungal PUFAs.

N-acetylcysteine in preclinical mouse and baboon models of thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic disorder diagnosed by thrombocytopenia and hemolytic anemia, associated with a deficiency in von Willebrand factor (VWF)-cleaving protease ADAMTS13. Current treatment is based on plasma infusion for congenital TTP, or plasma exchange, often in combination with immunosuppressive agents, for acquired TTP. These treatment methods are not always effective; therefore, new treatment methods are highly necessary. N-acetylcysteine (NAC), an FDA-approved anti-mucolytic agent, is a possible new treatment strategy for TTP, as it was demonstrated to reduce disulfide bonds in VWF, thereby decreasing VWF multimers size and hence their prothrombotic potential. We investigated whether NAC, without concurrent plasma exchange and immunosuppressive therapy, is effective in preventing and resolving TTP signs, using well-established murine and baboon models for TTP. In mice, prophylactic administration of NAC was effective in preventing severe TTP signs. This in vivo finding was supported by in vitro data demonstrating the VWF multimer-reducing properties of NAC in solution. Nonetheless, in both mice and baboons, administration of NAC was not effective in resolving preexisting TTP signs; thrombocytopenia, hemolytic anemia, and organ damage could not be reversed, as thrombus resolution was not achieved. Failure to improve clinical outcome occurred even though a reduction in VWF multimers was observed, demonstrating that NAC was efficient in reducing disulfide bonds in circulating VWF multimers. In conclusion, prophylactic administration of NAC, without concurrent plasma exchange, was effective in preventing severe TTP signs in mice, but NAC was not effective in resolving preexistent acute TTP signs in mice and baboons.

Comparison of common platelet receptors between the chacma baboon (Papio ursinus) and human for use in pre-clinical human-targeted anti-platelet studies

Abstract Anti-platelet agents play a central part in the treatment and prevention of acute thrombotic events. Discriminating animal models are needed for the development of novel agents. The chacma baboon has been extensively used as a model to evaluate anti-platelet agents. However, limited data exist to prove the translatability of this species to humans. We aimed to determine the suitability of the chacma baboon in preclinical human targeted GPIIb/IIIa, GPIbα and P2Y12 studies. Light-transmission platelet aggregometry (LTA), whole blood impedance aggregometry, receptor number quantification and genomic DNA sequencing were performed. Baboon ADP and arachidonic acid-induced LTA aggregation results differed significantly from human values, even at increased concentrations. LTA ristocetin-induced agglutination was comparable between species, but baboon platelets needed twice the concentration of ristocetin to elicit a similar response. Citrated baboon blood had significantly less aggregation than humans when evaluated with impedance aggregometry. However, hirudinised baboon whole blood gave similar aggregation as humans at the same agonist concentrations. GPIIb, GPIIIa and GPIbα numbers were significantly more on the baboon platelets. None of the amino acids deemed vital for receptor function, ligand binding or receptor inhibition, were radically different between the species. However, a conservative change in a calcium-binding region of GPIIb may render the baboon platelets more sensitive to calcium-binding agents. The chacma baboon may be used for the evaluation of human-targeted GPIIb/IIIa-, GPIbα- and P2Y12-inhibiting agents. However, the best anticoagulant, optimal agonist concentrations, increase in receptor number and sequence differences must be considered for any future studies.Anti-platelet agents play a central part in the treatment and prevention of acute thrombotic events. Discriminating animal models are needed for the development of novel agents. The chacma baboon has been extensively used as a model to evaluate anti-platelet agents. However, limited data exist to prove the translatability of this species to humans. We aimed to determine the suitability of the chacma baboon in preclinical human targeted GPIIb/IIIa, GPIbα and P2Y12 studies. Light-transmission platelet aggregometry (LTA), whole blood impedance aggregometry, receptor number quantification and genomic DNA sequencing were performed. Baboon ADP and arachidonic acid-induced LTA aggregation results differed significantly from human values, even at increased concentrations. LTA ristocetin-induced agglutination was comparable between species, but baboon platelets needed twice the concentration of ristocetin to elicit a similar response. Citrated baboon blood had significantly less aggregation than humans when evaluated with impedance aggregometry. However, hirudinised baboon whole blood gave similar aggregation as humans at the same agonist concentrations. GPIIb, GPIIIa and GPIbα numbers were significantly more on the baboon platelets. None of the amino acids deemed vital for receptor function, ligand binding or receptor inhibition, were radically different between the species. However, a conservative change in a calcium-binding region of GPIIb may render the baboon platelets more sensitive to calcium-binding agents. The chacma baboon may be used for the evaluation of human-targeted GPIIb/IIIa-, GPIbα- and P2Y12-inhibiting agents. However, the best anticoagulant, optimal agonist concentrations, increase in receptor number and sequence differences must be considered for any future studies.

The Cape Chacma baboon is not suitable for evaluating human targeted anti-GPVI agents.

Abstract An effective and safe anti-platelet drug is central to the management of patients with acute coronary syndrome (ACS). Glycoprotein VI (GPVI) is currently regarded as a potential target for novel anti-platelet agents due to its collagen-binding potential. Development of anti-thrombotics is associated with testing in animals. We have previously successfully evaluated anti-platelet drugs in the Cape Chacma baboon (Papio ursinus). However, various anti-GPVI agents did not have an effect on baboons when evaluated in our arterial thrombosis model. To evaluate the suitability of baboons for GPVI studies, we performed collagen-induced platelet aggregation, GPVI quantification and DNA sequencing. Baboon platelets needed double the amount of collagen compared to human platelets to illicit proper aggregation. GPVI quantification was unsuccessful due to non-binding of monoclonal antibodies. Sequencing of the GPVI gene revealed 36 SNPs leading to 14 amino acid changes, as well as a 9 bp deletion causing a 3 amino acid deletion. Several of the amino acid changes were within the ligand binding region of GPVI, causing limited binding of humanized anti-GPVI antibodies to the baboon platelets. Therefore, the baboon was deemed not suitable to evaluate human targeted anti-GPVI agents.

Tirofiban versus abciximab: tirofiban is administered at suboptimal dosages when evaluated in an arterial thrombosis model in non-human primates.

To prevent thrombosis in high-risk acute coronary syndrome patients undergoing percutaneous coronary intervention for re-vascularisation, concomitant administration of a glycoprotein IIb/IIIa inhibitor, such as abciximab, tirofiban or eptifibatide, is recommended. Abciximab and eptifibatide are mostly preferred over tirofiban, which is less effective in preventing ischaemic events. We compared the efficacy and bleeding potential of escalating doses of tirofiban and abciximab in non-human primates. The efficacy of tirofiban and abciximab in inhibiting cyclic flow reductions (CFRs) was tested in a high shear arterial thrombosis model. Bleeding was evaluated with the template bleeding time and an incision bleeding model. Abciximab completely inhibited arterial thrombosis after injection of its therapeutic bolus dose. With tirofiban, a dose three times higher than the recommended therapeutic dose caused weak inhibition characterised by a return of CFRs after re-injury. At nine times the recommended therapeutic dose, complete inhibition was observed, and the efficacy of tirofiban was comparable to abciximab at its therapeutic bolus dose. Blood loss was less than with abciximab at its effective dose. In this model, tirofiban compared favourably with abciximab, although only at a dose of 3–9 times the therapeutic dose, and caused less bleeding than abciximab.

Lifestyle Change Alone Sufficient to Lower Cholesterol in Male Patient With Moderately Elevated Cholesterol: A Case Report

Background. Cardiovascular disease is a major cause of deaths. Elevated cholesterol levels to above the normal reference range is a major risk factor for developing cardiovascular disease. Current guidelines recommend the use of cholesterol-lowering drugs to lower cholesterol levels to within the normal reference range. However, the American Heart Association further recommends a change in lifestyle in managing cholesterol levels. Thus, cholesterol-lowering drugs may not be needed if a lifestyle-change alone is sufficient in lowering cholesterol levels to within normal ranges. Unfortunately, limited examples exist in academic literature to illustrate the effectiveness of lifestyle change alone in lowering of cholesterol levels. Case report. We report a case of a 33-year-old man, with moderately elevated cholesterol levels and a family history of cardiovascular disease. Method. The man followed an altered healthy fat diet accompanied with moderate exercise for 6 weeks, without the addition of cholesterol-lowering agents. Results. At the 6-week follow-up, he was able to decrease his total cholesterol by 40.25% and low-density lipid cholesterol by 52.8%, to within normal ranges. The cholesterol levels remained within normal ranges after 6 months. Conclusion. This case illustrates that in some individuals, lifestyle change alone is sufficient to lower moderately elevated cholesterol levels.Background. Cardiovascular disease is a major cause of deaths. Elevated cholesterol levels to above the normal reference range is a major risk factor for developing cardiovascular disease. Current guidelines recommend the use of cholesterol-lowering drugs to lower cholesterol levels to within the normal reference range. However, the American Heart Association further recommends a change in lifestyle in managing cholesterol levels. Thus, cholesterol-lowering drugs may not be needed if a lifestyle-change alone is sufficient in lowering cholesterol levels to within normal ranges. Unfortunately, limited examples exist in academic literature to illustrate the effectiveness of lifestyle change alone in lowering of cholesterol levels. Case report. We report a case of a 33-year-old man, with moderately elevated cholesterol levels and a family history of cardiovascular disease. Method. The man followed an altered healthy fat diet accompanied with moderate exercise for 6 weeks, without the addition of cholesterol-lowering agents. Results. At the 6-week follow-up, he was able to decrease his total cholesterol by 40.25% and low-density lipid cholesterol by 52.8%, to within normal ranges. The cholesterol levels remained within normal ranges after 6 months. Conclusion. This case illustrates that in some individuals, lifestyle change alone is sufficient to lower moderately elevated cholesterol levels.

Comparison of Commercially Available Blood Collection Tubes Containing Sodium Citrate and Hirudin in Platelet Aggregation Testing

BACKGROUND Platelet reactivity assessment is an important tool in both the causal determination of bleeding diathesis as well as in the evaluation of the efficacy of anti-platelet therapy in patients at risk of thrombosis. Sodium citrate is the most widely used anticoagulant for hemostasis investigations. However, some doubt exists over the suitability of sodium citrate in platelet function testing. Hirudin has been suggested as a superior replacement. Nevertheless, only 1 study compared citrated and hirudin treated samples with light transmission aggregometry. Therefore, limited evidence exists to conclusively prove the supremacy of hirudin over sodium citrate in light transmission aggregometry. The aim of our study was to compare citrated and hirudin treated samples, collected in commercially available blood collection tubes, using the 5 most common agonists, with light transmission aggregometry. MATERIAL AND METHODS Blood was obtained from 20 healthy volunteers. Platelet counts were performed on platelet-rich plasma. Light transmission aggregometry was performed within 4 h of sample collection using ADP, collagen, arachidonic acid, epinephrine, and ristocetin as agonists. RESULTS Platelet counts for the respective anticoagulants did not differ significantly. ADP-induced aggregation was comparable between the samples. However, among all the agonists, hirudin-treated platelets had significantly weaker aggregatory responses. CONCLUSIONS Commercially available sodium citrate should remain the anticoagulant of choice for routine platelet function testing in our setting. However, the time limitation associated with the use of sodium citrate in platelet function testing remains a concern. Thus, alternative anticoagulants should still be explored.Background Platelet reactivity assessment is an important tool in both the causal determination of bleeding diathesis as well as in the evaluation of the efficacy of anti-platelet therapy in patients at risk of thrombosis. Sodium citrate is the most widely used anticoagulant for hemostasis investigations. However, some doubt exists over the suitability of sodium citrate in platelet function testing. Hirudin has been suggested as a superior replacement. Nevertheless, only 1 study compared citrated and hirudin treated samples with light transmission aggregometry. Therefore, limited evidence exists to conclusively prove the supremacy of hirudin over sodium citrate in light transmission aggregometry. The aim of our study was to compare citrated and hirudin treated samples, collected in commercially available blood collection tubes, using the 5 most common agonists, with light transmission aggregometry. Material/Methods Blood was obtained from 20 healthy volunteers. Platelet counts were performed on platelet-rich plasma. Light transmission aggregometry was performed within 4 h of sample collection using ADP, collagen, arachidonic acid, epinephrine, and ristocetin as agonists. Results Platelet counts for the respective anticoagulants did not differ significantly. ADP-induced aggregation was comparable between the samples. However, among all the agonists, hirudin-treated platelets had significantly weaker aggregatory responses. Conclusions Commercially available sodium citrate should remain the anticoagulant of choice for routine platelet function testing in our setting. However, the time limitation associated with the use of sodium citrate in platelet function testing remains a concern. Thus, alternative anticoagulants should still be explored.