In vivo evaluation of monoclonal antibody M4M using a humanised rat model of stroke demonstrates attenuation of reperfusion injury via blocking human TRPM4 channel.

BACKGROUND: Blocking Transient Receptor Potential Melastatin 4 (TRPM4) in rodents by our antibody M4P has shown to attenuate cerebral ischaemia-reperfusion injury. Since M4P does not interact with human TRPM4, the therapeutic potential of blocking human TRPM4 remains unclear. We developed a monoclonal antibody M4M that inhibited human TRPM4 in cultured cells. However, M4M has no effect on stroke outcome in wild-type rats. Therefore, M4M needs to be evaluated on animal models expressing human TRPM4. METHODS: We generated a humanised rat model using the CRISPR/Cas technique to knock-in (KI) the human TRPM4 antigen sequence. RESULTS: In primary neurons from human TRPM4 KI rats, M4M binds to hypoxic neurons, but not normoxic nor wild-type neurons. Electrophysiological studies showed that M4M blocked ATP depletion-induced activation of TRPM4 and inhibited hypoxia-associated cell volume increase. In a stroke model, administration of M4M reduced infarct volume in KI rats. Rotarod test and Neurological deficit score revealed improvement following M4M treatment. CONCLUSION: M4M selectively binds and inhibits hypoxia-induced human TRPM4 channel activation in neurons from the humanised rat model, with no effect on healthy neurons. Use of M4M in stroke rats showed functional improvements, suggesting the potential for anti-human TRPM4 antibodies in treating acute ischaemic stroke patients.

Ex vivo haemostatic capacity of plasma upon thawing and beyond: a comparison between fresh frozen plasma (FFP) and frozen plasma prepared from whole blood stored at room temperature up to 24 hours postcollection (RTFP24).

BACKGROUND/OBJECTIVES: We compared the ex vivo haemostatic capacity of RTFP24 with FFP upon thawing and >24 h post-thaw. We included thrombin generation (TG) as few studies had compared global haemostatic function, and most did not directly compare RTFP24 with FFP >24 h post-thaw. MATERIALS/METHODS: Twenty units each of RTFP24 and FFP were measured for coagulation factors and thrombin generation upon thawing (D0) and 4 days post-thaw (D4). Labile factors were also measured from D1 to D3. 10 single cryoprecipitate units were each prepared from FFP and RTFP24, and measured for FXIII, FVIII and fibrinogen at D0. RESULTS: At D0, RTFP24 was comparable to FFP except for lower FV, protein S, endogenous thrombin potential (ETP) and higher FXIII. These differences were likely clinically insignificant since 95% and 80% of RTFP24 met our laboratory's reference ranges for FV/protein S and ETP, respectively. There were no differences between RTFP24- and FFP-derived cryoprecipitate. At D4, RTFP24 was comparable to FFP except for lower FV, ETP, and higher FXI and FXIII. More RTFP24 than FFP had ETP lower than our laboratory's reference range (45% vs 15%). Multiple coagulation factors and all TG parameters declined from their respective baselines. The percentage declines were comparable or less in RTFP24, except for protein C, fibrinogen and time to peak. CONCLUSION: RTFP24 and FFP, and their derived cryoprecipitate have comparable haemostatic capacity upon thawing. RTFP24 has poorer TG potential than FFP >24 h post-thaw, not supporting universal extension of RTFP24's shelf life except to facilitate urgent transfusions for massive haemorrhage.