Activated protein C–protein C inhibitor

Activated protein C–protein C inhibitor (APC-PCI) is a complex of activated protein C (APC) and protein C inhibitor (PCI). It has been measured in coagulation testing to evaluate coagulation, thrombosis, and other cardiovascular complications. It is a marker of thrombin generation and indicates hypercoagulability. The half-life of APC-PCI is either 40 minutes or 140 minutes.

Ethinylestradiol-containing birth control pills have been found to increase levels of APC-PCI to a similar degree as thrombin–antithrombin complex (TAT) and to a greater extent than D-dimer. However, only APC-PCI was able to differentiate between a second- and third-generation birth control pill.

Another complex related to APC-PCI is the activated protein C–α₁-antitrypsin (APCAT) complex.

Biochemical Properties

Protein C Inhibitor is a glycoprotein with a molecular weight of approximately 54 Kilodaltons (kDa). It belongs to the serpin superfamily, characterized by their unique inhibitory mechanism involving a reactive center loop (RCL). Upon interaction with APC, PCI forms a stable, covalent complex with a total molecular weight of around 96 kDa. This complex is rapidly formed in plasma and is considered one of the primary means by which APC activity is neutralized.

The N-terminal sequence of PCI begins with Gly-Arg-Thr-Cys-Pro-Lys-Pro-Asp, which is indicative of its structural similarity to other serpins, including trypsin inhibitors. The glycosylation status of PCI influences its stability, half-life, and interaction with target proteases. The stability and rapid formation of the APC–PCI complex make it a reliable marker in the evaluation of in vivo APC activity.

Mechanism of Inhibition

The inhibition of activated protein C by PCI occurs through a serpin-class suicide inhibition mechanism. The reactive center loop of PCI acts as a decoy substrate, inserting into the active site of APC. Once cleaved by APC, PCI undergoes a significant conformational change that traps the enzyme in a covalently bonded, inactive state. This 1:1 stoichiometry binding permanently neutralizes APC, thus terminating its anticoagulant and cytoprotective actions.

This regulatory mechanism is crucial in controlling coagulation. While APC functions to inactivate procoagulant factors Va and VIIIa, thereby preventing thrombin formation, unchecked activity of APC could potentially lead to bleeding disorders. PCI ensures that APC is tightly regulated, maintaining a delicate balance between clot formation and inhibition.

Marker of Coagulation Activation

The APC–PCI complex serves as a sensitive and specific biomarker for activated coagulation, particularly the Protein C pathway. Elevated levels of the complex are observed in a range of Thrombosis conditions such as Deep vein thrombosis (DVT), pulmonary embolism (PE), and disseminated intravascular coagulation (DIC). Measuring APC–PCI levels provides insight into the real-time activation of the anticoagulant pathway and its inhibition, making it a valuable tool in clinical diagnostics.

Oral Contraceptives and APC–PCI

Studies have shown that women using third-generation combined oral contraceptive pill (COCs) have significantly elevated levels of APC–PCI compared to those using second-generation formulations. This increase in APC–PCI formation corresponds to higher thrombotic risk. Notably, APC–PCI has demonstrated greater discriminatory power than D-dimer in assessing thrombogenic differences between these contraceptives, indicating its potential utility in evaluating hormone-induced coagulopathy.

Coagulation Factor Deficiencies

Interestingly, individuals with combined Factor V and Factor VIII deficiency—a rare autosomal recessive bleeding disorder—have been shown to maintain normal levels of functional APC–PCI complexes. Interestingly, individuals with combined Factor V and Factor VIII deficiency—a rare autosomal recessive bleeding disorder—have been shown to maintain normal levels of functional APC–PCI complexes. This was highlighted in a seminal study which provided strong evidence that PCI’s inhibitory function is not significantly impaired in the presence of deficient cofactors required for Protein C activation. This suggests that PCI activity is robust and independent of upstream coagulation cascade variability.

Diagnostic and Research Applications

Due to its specificity and stability, APC–PCI is frequently employed in clinical research and diagnostic testing. Elevated plasma levels indicate ongoing thrombin generation and counter-regulatory anticoagulant responses. It is particularly useful in:

• Monitoring hypercoagulable states post-surgery, trauma, or during pregnancy.

• Evaluating the efficacy of anticoagulant treatments.

• Investigating the pathophysiology of inherited or acquired coagulation disorders.

When used in conjunction with other markers like thrombin-antithrombin (TAT) complexes, D-dimer, and prothrombin fragment 1+2 (F1+2), APC–PCI provides a comprehensive picture of hemostatic activity and endothelial function.

Comparative Biology and Homology

Protein C Inhibitor shares structural and sequence homology (biology) with other members of the serpin family, such as α1-antitrypsin, antithrombin, and tissue factor pathway inhibitor (TFPI). In particular, the N-terminal sequence of PCI exhibits similarity to the bovine colostrum trypsin inhibitor and the human pancreatic secretory trypsin inhibitor, indicating evolutionary conservation of its inhibitory domain. This cross-species homology underscores the conserved nature of serpin function in regulating proteolytic cascades, especially in coagulation, inflammation, and fertility.

Associated Complexes in Plasma

While PCI is a primary inhibitor of activated protein C, other plasma proteins can also form inhibitory complexes with APC:

• APC–α1-antitrypsin (APC–AT) : Formed less rapidly than APC–PCI but also contributes to the regulation of APC activity.

• APC–α2-macroglobulin : Another alternative inhibitory complex, although less prominent in physiological conditions.

However, due to its rapid formation and stability, the APC–PCI complex is considered the most reliable surrogate marker for evaluating APC.