The lectin pathway of complement activation is an important component of the innate immune defense. The initiation complexes of the lectin pathway consist of a recognition molecule and associated serine proteases. Until now the autoactivating mannose-binding lectin-associated serine protease (MASP)-2 has been considered the autonomous initiator of the proteolytic cascade. The role of the much more abundant MASP-1 protease was controversial. Using unique, monospecific inhibitors against MASP-1 and MASP-2, we corrected the mechanism of lectin-pathway activation. In normal human serum, MASP-2 activation strictly depends on MASP-1. MASP-1 activates MASP-2 and, moreover, inhibition of MASP-1 prevents autoactivation of MASP-2. Furthermore we demonstrated that MASP-1 produces 60% of C2a responsible for C3 convertase formation.

MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways.

Objective— Coated-platelets are a subset of cells observed during costimulation of platelets with collagen and thrombin. Important characteristics of coated-platelets include retention of multiple α-granule proteins and expression of phosphatidylserine on the cell surface. The mitochondrial permeability transition pore (MPTP) is a key step in apoptosis and is suggested to be involved in some forms of platelet activation. The objective of this study was to examine the role of MPTP in the synthesis of coated-platelets. Methods and Results— Flow cytometric analysis of coated-platelet production was used to examine the impact of pharmacological effectors of MPTP formation. Cyclosporin A, coenzyme Q, and bongkrekic acid all inhibit MPTP formation as well as production of coated-platelets. Phenylarsine oxide and diamide, both potentiators of MPTP formation, stimulate coated-platelet synthesis. Atractyloside, another inducer of MPTP formation, does not affect the percentage of coated-platelets synthesized; however, it does increase the level of phosphatidylserine exposed on the surface of coated-platelets. Conclusions— These findings indicate that MPTP formation is an integral event in the synthesis of coated-platelets. Although the exact function of the MPTP remains to be determined, these data support a growing body of evidence that apoptosis-associated events are vital components of the platelet activation process.

The complement system, an essential part of the innate immune system, can be activated through three distinct routes: the classical, the alternative, and the lectin pathways. The contribution of individual activation pathways to different biological processes can be assessed by using pathway-selective inhibitors. In this paper, we report lectin pathway-specific short peptide inhibitors developed by phage display against mannose-binding lectin-associated serine proteases (MASPs), MASP-1 and MASP-2. On the basis of the selected peptide sequences, two 14-mer peptides, designated as sunflower MASP inhibitor (SFMI)-1 and SFMI-2, were produced and characterized. SFMI-1 inhibits both MASP-1 and MASP-2 with a K_I of 65 and 1030 nM, respectively, whereas SFMI-2 inhibits only MASP-2 with a K_I of 180 nM. Both peptides block the lectin pathway activation completely while leaving the classical and the alternative routes intact and fully functional, demonstrating that of all complement proteases only MASP-1 and/or MASP-2 are inhibited by these peptides. In a C4 deposition inhibitor assay using preactivated MASP-2, SFMI-2 is 10-fold more effective than SFMI-1 in accordance with the fact that SFMI-2 is a more potent inhibitor of MASP-2. Surprisingly, however, out of the two peptides, SFMI-1 is much more effective in preventing C3 and C4 deposition when normal human serum containing zymogen MASPs is used. This suggests that MASP-1 has a crucial role in the initiation steps of lectin pathway activation most probably by activating MASP-2. Because the lectin pathway has been implicated in several life-threatening pathological states, these inhibitors should be considered as lead compounds toward developing lectin pathway blocking therapeutics.

Activation of platelets with 2 agonists, collagen and thrombin, reveals a subpopulation of cells referred to as COAT-platelets (collagen and thrombin activated). These cells are enriched in several membrane-bound, procoagulant proteins, including fibrinogen, thrombospondin, factor V, von Willebrand factor, and fibronectin. α-Granule proteins bound to COAT-platelets are derivatized with serotonin by a transglutaminase-mediated process, and the interaction of conjugated serotonins with unidentified serotonin binding sites on the platelet surface enhances retention of these proteins. We now demonstrate that both thrombospondin and fibrinogen provide the requisite serotonin binding sites. Thrombospondin and fibrinogen were identified using photoreactive cross-linking to an albumin-(serotonin)6conjugate during COAT-platelet production. We subsequently verified that biotin-albumin-(serotonin)6 binds in vitro to thrombospondin, fibrinogen, and fibrinogen fragment D in a saturable manner. These data support a model for COAT-platelets where serotonin-derivatized procoagulant proteins interact with their respective receptors (eg, fibrinogen with glycoprotein IIb/IIIa or factor V with phosphatidylserine) as well as serotonin binding sites on fibrinogen and thrombospondin, resulting in a stable, multivalent complex on the cell surface.

COAT platelets are a recently described subpopulation of cells resulting from simultaneous activation with collagen and thrombin. The complete process by which COAT platelets are produced is still not clear, although significant recent progress has been made. COAT platelets retain several procoagulant proteins on their surface by a previously unrecognized mechanism involving transglutaminase mediated conjugation of serotonin to released alpha-granule proteins. Fibrinogen and thrombospondin have been found to bind serotonin-conjugated proteins and thereby provide the requisite link for stabilization of serotonin-derivatized, procoagulant proteins on COAT platelets. Multivalent interactions, resulting from traditional receptor interactions and binding of conjugated serotonin by fibrinogen and thrombospondin, result in exceptionally strong retention of procoagulant alpha-granule proteins on the surface of COAT platelets. The physiologic significance of this new subclass of platelets remains to be determined.