A family with a history of recurring thrombosis was studied to determine if a plasma protein deficiency could account for the observed disease. Protein C levels in plasma were determined immunologically using the Laurell rocket technique. The propositus, his father, and his paternal uncle, who are severely affected, had 38-49% of normal levels of protein C antigen, whereas unaffected family members had normal levels. There was no familial deficiency of antithrombin III and plasminogen. Because activated protein C is a potent in vitro anticoagulant enzyme and an in vivo profibrinolytic agent, it is suggested that the recurrent thrombotic disease in this family is due to an inherited deficiency in protein C.

Protein C is best known for its mild deficiency associated with venous thrombosis risk and severe deficiency associated with neonatal purpura fulminans. Activated protein C (APC) anticoagulant activity involves proteolytic inactivation of factors Va and VIIIa, and APC resistance is often caused by factor V Leiden. Less known is the clinical success of APC in reducing mortality in severe sepsis patients (PROWESS trial) that gave impetus to new directions for basic and preclinical research on APC. This review summarizes insights gleaned from recent in vitro and in vivo studies of the direct cytoprotective effects of APC that include beneficial alterations in gene expression profiles, anti-inflammatory actions, antiapoptotic activities, and stabilization of endothelial barriers. APC's cytoprotection requires its receptor, endothelial cell protein C receptor, and protease-activated receptor-1. Because of its pleiotropic activities, APC has potential roles in the treatment of complex disorders, including sepsis, thrombosis, and ischemic stroke. Although much about molecular mechanisms for APC's effects on cells remains unclear, it is clear that APC's structural features mediating anticoagulant actions and related bleeding risks are distinct from those mediating cytoprotective actions, suggesting the possibility of developing APC variants with an improved profile for the ratio of cytoprotective to anticoagulant actions.

The total chemical synthesis of proteins has great potential for increasing our understanding of the molecular basis of protein function. The introduction of native chemical ligation techniques to join unprotected peptides next to a cysteine residue has greatly facilitated the synthesis of proteins of moderate size. Here, we describe a straightforward methodology that has enabled us to rapidly analyze the compatibility of the native chemical ligation strategy for X–Cys ligation sites, where X is any of the 20 naturally occurring amino acids. The simplified methodology avoids the necessity of specific amino acid thioester linkers or alkylation of C-terminal thioacid peptides. Experiments using matrix-assisted laser-desorption ionization MS analysis of combinatorial ligations of LYRAX-C-terminal thioester peptides to the peptide CRANK show that all 20 amino acids are suitable for ligation, with Val, Ile, and Pro representing less favorable choices because of slow ligation rates. To illustrate the method’s utility, two 124-aa proteins were manually synthesized by using a three-step, four-piece ligation to yield a fully active human secretory phospholipase A₂and a catalytically inactive analog. The combination of flexibility in design with general access because of simplified methodology broadens the applicability and versatility of chemical protein synthesis.

It is well recognized that high-density lipoprotein (HDL)-cholesterol is antiatherogenic and serves a role in mediating cholesterol efflux from cells. However, HDL has multiple additional endothelial and antithrombotic actions that may also afford cardiovascular protection. HDL promotes the production of the atheroprotective signaling molecule nitric oxide (NO) by upregulating endothelial NO synthase (eNOS) expression, by maintaining the lipid environment in caveolae where eNOS is colocalized with partner signaling molecules, and by stimulating eNOS as a result of kinase cascade activation by the high-affinity HDL receptor scavenger receptor class B type I (SR-BI). HDL also protects endothelial cells from apoptosis and promotes their growth and their migration via SR-BI–initiated signaling. As importantly, there is evidence of a variety of mechanisms by which HDL is antithrombotic and thereby protective against arterial and venous thrombosis, including through the activation of prostacyclin synthesis. The antithrombotic properties may also be related to the abilities of HDL to attenuate the expression of tissue factor and selectins, to downregulate thrombin generation via the protein C pathway, and to directly and indirectly blunt platelet activation. Thus, in addition to its cholesterol-transporting properties, HDL favorably regulates endothelial cell phenotype and reduces the risk of thrombosis. With further investigation and resulting greater depth of understanding, these mechanisms may be harnessed to provide new prophylactic and therapeutic strategies to combat atherosclerosis and thrombotic disorders.