The influence of the type of intracranial lesion on the final outcome in a consecutive series of 277 severely head-injured patients was analyzed. Patients were studied with computerized tomography (CT) and underwent continuous measurement of intracranial pressure. They received identical treatment according to a standardized protocol. Outcome of patients with either epidural hematoma (38 cases), subdural hematoma (56 cases), brain contusion (87 cases), or diffuse brain damage (96 cases) was rather heterogeneous, and serial CT scanning allowed the authors to outline eight consistent anatomical patterns in the whole series which have stronger prognostic significance than the four major lesion categories mentioned above. Patients with pure extracerebral hematoma (19 cases), single brain contusion (45 cases), general brain swelling (41 cases), and normal CT scans (28 cases) had a significantly better outcome than patients developing acute hemispheric swelling after operation for a large extracerebral hematoma (27 cases), patients with multiple brain contusion, either unilateral or bilateral (74 cases), and patients with diffuse axonal injury (43 cases). These anatomical patterns are interesting because, in addition to having clinical and physiopathological significance, they provide useful prognostic information and facilitate improved therapeutic decision-making in severely head-injured patients.

The vertebrate lens behaves like a syncytium, and it is formed mainly by cells called lens fibers. Between the fibers are extensive networks of membrane junctions. The major intrinsic protein (MIP) constitutes about 50-60% of the intrinsic membrane proteins found in lens fiber junctions. The role of MIP is unknown. Nevertheless, it has been proposed that it is the protein responsible for the adhesion between the plasmatic membranes of the lens fibers. The aim of our studies was to test the adhesion-promoting role of MIP. We reconstituted MIP into large unilamellar vesicles (LUV) of phosphatidylcholine (PC) and studied the vesicle aggregation between MIP-reconstituted LUV (PC-MIP) and phosphatidylserine (PS) vesicles. The aggregation process was monitored using methods based on resonance energy transfer (RET) and turbidity measurements. Neither RET nor an increase in turbidity occurred in any combination except in the presence of both MIP and PS. The liposomes thus aggregate through protein-lipid interactions. These results show that MIP promotes adhesion with negatively charged membranes, indicating that the adhesion is electrostatic in nature. Aggregation was fastest at pH 6.0. The aggregation effect was abolished with pronase treatment. Preincubation of PC-MIP vesicles with anti-MIP polyclonal serum also inhibited the aggregation. These studies are the first experimental evidence supporting the hypothesis of an adhesive role for MIP.

Treatment of bleeding disorders using transfusion of donor-derived platelets faces logistical challenges due to their limited availability, high risk of contamination, and short (5 to 7 days) shelf life. These challenges could be potentially addressed by designing platelet mimetics that emulate the adhesion, aggregation, and procoagulant functions of platelets. To this end, we created liposome-based platelet-mimicking procoagulant nanoparticles (PPNs) that can expose the phospholipid phosphatidylserine on their surface in response to plasmin. First, we tested PPNs in vitro using human plasma and demonstrated plasmin-triggered exposure of phosphatidylserine and the resultant assembly of coagulation factors on the PPN surface. We also showed that this phosphatidylserine exposed on the PPN surface could restore and enhance thrombin generation and fibrin formation in human plasma depleted of platelets. In human plasma and whole blood in vitro, PPNs improved fibrin stability and clot robustness in a fibrinolytic environment. We then tested PPNs in vivo in a mouse model of thrombocytopenia where treatment with PPNs reduced blood loss in a manner comparable to treatment with syngeneic platelets. Furthermore, in rat and mouse models of traumatic hemorrhage, treatment with PPNs substantially reduced bleeding and improved survival. No sign of systemic or off-target thrombotic risks was observed in the animal studies. These findings demonstrate the potential of PPNs as a platelet surrogate that should be further investigated for the management of bleeding.

Protease activated receptor 4 (PAR4) mediates sustained thrombin signaling in platelets and is required for a stable thrombus. PAR4 is activated by proteolysis of the N-terminus to expose a tethered ligand. The structural basis for PAR4 activation and the location of its ligand binding site (LBS) are unknown. Using hydrogen/deuterium exchange (H/D exchange), computational modeling and signaling studies, we determined the molecular mechanism for tethered ligand-mediated PAR4 activation. H/D exchange identified that the LBS is composed by transmembrane domain 3 (TM3) and TM7. Unbiased computational modeling further predicted an interaction between Gly48 from the tethered ligand and Thr153 from the LBS. Mutating Thr153 significantly decreased PAR4 signaling. H/D exchange and modeling also showed that extracellular loop 3 (ECL3) serves as a gatekeeper for the interaction between the tethered ligand and LBS. A naturally occurring sequence variant (P310L, rs2227376) and two experimental mutations (S311A and P312L) determined that the rigidity conferred by prolines in ECL3 are essential for PAR4 activation. Finally, we examined the role of the polymorphism at position 310 in venous thromboembolism (VTE) using the INVENT consortium multi-ancestry GWAS meta-analysis. Individuals with the PAR4 Leu310 allele had a 15% relative risk reduction for VTE (odds ratio [OR]: 0.85; 95% CI: 0.77-0.94) compared to the Pro310 allele. These data are consistent with our H/D exchange, molecular modeling, and signaling studies. In conclusion, we have uncovered the structural basis for PAR4 activation and identified a previously unrecognized role for PAR4 in VTE.