Compound heterozygous protein C variants undetectable by common laboratory testing causing purpura fulminans after the neonatal period
Authors: Kyle L. MacQuarrie, Olatundun Williams, Kenneth D. Friedman, Rachel S. Bercovitz
Journal: American Journal of Hematology (2020)

The patient, a four-year-old female, presents to the Emergency Department (ED) with acute onset of bilateral lower extremity and back bruising (Figure 1A,B). Her parents deny any recent trauma or infectious symptoms. Her history is significant for a perinatal stroke in the setting of an unremarkable pregnancy and delivery, an additional stroke due to a sinus venous thrombosis at age two, global developmental delay, and blindness. Prior hereditary thrombophilia work-up, including factor V Leiden, had been performed and was negative, but she had been given a tentative diagnosis of dysfibrinogenemia based on prior episodes of significant bruising and intermittent hypofibrinogenemia on prior coagulation testing. In the previous 2 years, she has had multiple episodes of bruising significant enough to prompt urgent evaluation; these episodes lasted between 2 and 24 hours. Previous episodes presented with ecchymoses in patterns consistent with possible injury from pressure points on her wheelchair. One of these episodes presented with laboratory tests (labs) consistent with a consumptive coagulopathy, but as her lesions faded, her labs normalized without intervention. Due to this history and progression of the lesions while in the ED, blood counts and coagulation labs were obtained. Her initial laboratory evaluation was notable for multiple coagulation abnormalities, with a prothrombin time (PT) >100 seconds (reference range 9.3-12.0 seconds), activated partial thromboplastin time (aPTT) >120 seconds (reference range 23.6-30.7 seconds), fibrinogen <70 mg dL−1 (reference range 180-410 mg dL−1) and an elevated D-dimer at 4.55 μg mL−1 (reference range <0.65 μg mL−1). She had mild thrombocytopenia (110 × 109 L−1, reference range 150-450 × 109 L−1), and her hemoglobin and white blood cell counts were within the normal range for her age. Her labs at this time were concerning for a consumptive process, with a severe hypofibrinogenemia lower than her baseline levels and new thrombocytopenia, in addition to the severely prolonged PT and aPTT. However, apart from the ecchymoses, she was not noted to have any evidence of oozing or bleeding on her initial evaluation. Given her lack of any bleeding symptoms, it was decided not to administer any blood products and instead observe closely, as there was concern that blood products might increase her risk of thrombosis. During this ED evaluation, her skin discoloration was noted to be spreading, and she was admitted to the intensive care unit (ICU) for observation. Over the next 24 hours, her providers and parents noted the lesions diminished in size without any specific treatment. Her PT and aPTT both improved (16.1 and 49.0 seconds, respectively), though her D-dimer rose to >22.0 μg mL−1. Given the rapid improvement in her skin lesions, many of them diminishing notably in size over the course of a single night, the clinical team were unsure whether they truly represented ecchymoses, or if they were due to a vasculitic process that was resulting in consumption and coagulation abnormalities. Similar to her previous episodes, she had improved over a short time period without any intervention. Her coagulation labs improved by the following day (PT 11.9 seconds, PTT 35.8 seconds, fibrinogen 154 mg dL−1, D-dimer 11.21 μg mL−1), and providers noted complete resolution of a subset of her skin lesions, though one of the largest lesions began to take on a more necrotic appearance in the central region (Figure 1C). With this improvement, she was transferred out of the ICU. Soon after, she developed low-grade fevers and was started on antibiotic coverage for presumed gram-positive bacteria. While it was reassuring that her coagulation abnormalities improved, the necrotic appearance of a lesion was concerning and puzzling, as such a development is not typical of hypofibrinogenemia or dysfibrinogenemia. Anti-coagulation was considered at this time but not started due to the unclear etiology of her underlying process and the risk of potential worsening of her skin necrosis. In discussion with both dermatology and plastic surgery, it was decided that a skin biopsy of the most prominent lesion would be performed when her coagulation labs showed further improvement. With the new onset of fevers and given her developing skin breakdown, gram-positive bacteria were deemed to be the most likely infectious agents. Over the next 12 hours, multiple purpuric lesions re-emerged on her lower extremities and back and a subset of these lesions also started to take on a necrotic appearance. She was transferred back to the ICU for empiric, multi-faceted treatment. Her labs demonstrated worsening thrombocytopenia (63 × 109 L−1), PT (15.0 seconds), fibrinogen (105 mg dL−1), aPTT (>120 seconds) and D-dimer (>22.0 μg mL−1), and schistocytes were now noted on peripheral blood smears, though she had no evidence of increased lactate or worsening kidney or liver function. Systemic anti-coagulation with unfractionated heparin was started, and steroids (2 mg/kg/day of methylprednisolone), fresh frozen plasma (FFP), and broad-spectrum antibiotics were all administered. Note, CT/CTA imaging of her chest, pelvis and lower extremities was performed, but did not identify an occult thrombus. Given the rapid progression of her symptoms, and the significant worsening of her laboratory results, aggressive empiric therapy was initiated. The differential at that point in time was broad and included a thrombotic microangiopathy (TMA), cryoglobulinemia, vasculitis, dysfibrinogenemia, and disseminated intravascular coagulopathy (DIC). Contrary to a typical presentation of DIC, she had no oozing or bleeding at venipuncture sites. Her schistocytes and thrombocytopenia were concerning for TMA. Note, DIC is a secondary process, but no primary process had thus far been elucidated. Her apparent lack of arterial or venous thrombus was also perplexing if, in fact, her skin lesions were secondary to a diffuse pro-thrombotic state. Plasmapheresis with FFP was initiated shortly after steroids and antibiotics were begun. Even with central venous line placement, there were no issues with bleeding. She received a total of four daily plasmapheresis treatments and remained on therapeutic anti-coagulation. Her purpura remained stable in extent and appearance during that time. Her PT and fibrinogen normalized (11.8 seconds and 244 mg dL−1, respectively), and her D-dimer improved (1.15 μg mL−1) by the fourth day. Blood cultures remained negative. One of the lesions was biopsied and the pathology was consistent with a thrombotic vaso-occlusive process, with no evidence of vasculitis. Her lab evaluation was notable for a normal protein C activity by chromogenic assay (86 IU dL−1, laboratory reference range: 45-160, Berichrom Protein C, Siemens), as well as normal protein S activity (87 IU dL−1, laboratory reference range: 55-125); both tested samples were drawn prior to administration of any plasma. Age-specific published reference range for protein C activity in children aged 1-5 years is 40-92 IU dL−1, which is lower than the adult range of 64-128 IU dL−1 published in the same manuscript.1 The patient had a qualitatively positive cryoglobulin serum assay with very low quantitation (<1% cryocrit). Plasmapheresis had been started empirically as it is used to treat TMA, cryoglobulinemia, and vasculitis2 and therefore addressed multiple potential etiologies. Interpretation of her clinical improvement was confounded by her receipt of multiple treatments simultaneously. Whether her improvement was a result of the plasmapheresis, the steroid burst, and/or anti-coagulation was unclear. The positive qualitative result for cryoglobulins was the first test result that suggested a potential etiology, but the very low quantitation was inconsistent with such a significant episode of purpura fulminans. While the pathology results of her skin biopsy ruled out a vasculitis as the underlying cause, it did not rule in a cause. Her lesions stabilized after the plasmapheresis was stopped, with a slow sloughing of the necrotic areas that was treated symptomatically. Her steroids were weaned gradually, and her anti-coagulation was changed from therapeutic to prophylactic dosing while she remained inpatient. She continued to improve clinically even after these treatment changes and was discharged from the hospital on low-molecular-weight heparin (LMWH) with a goal anti-Xa peak of 0.2-0.5 IU/mL. Following discharge, the patient returned to her usual state of health and has continued prophylactic LMWH. She has had no further lesions or complications following discharge. While she had clinically improved, no definitive cause of this episode was identified. Given the severity of her course, there was concern that she had an unidentified hypercoagulability disorder. The clinical team decided to pursue a genetic workup. Whole exome sequencing was performed on the patient. She was found to be a compound heterozygote inheriting variant PROC (protein C) genes from both parents. The patient and her mother share a three-nucleotide deletion affecting glutamine at position 154 (c.461_463delAGG) in exon 6 of the PROC gene that has previously been described in the heterozygous state in an individual with quantitative protein C deficiency and a history of thrombi.3 The patient and her father share a single nucleotide variant (p.Arg42His) in exon 3 of the PROC gene (ie, pR42H variant) that results in an amino acid substitution previously described in a familial cohort with protein C deficiency and a history of thrombi.4 Based on the genetic results, she continued her prophylactic LMWH indefinitely to reduce risk of future thrombotic episodes. The p.R42H variant is associated with discrepant results between the commonly performed amidolytic assay, which uses degradation of a chromogenic substrate as an endpoint, and the clot-based assay, which reflects degradation of activated factor V (Va) or activated factor VIII (VIIIa).4 This discrepancy suggested a potential explanation for the patient's normal protein C testing results in the past. Given the patient's young age, as well as her history of a highly variable diet and difficulties with venipuncture due to her developmental delays, it was decided to continue her on LMWH, as opposed to utilizing anticoagulants such as warfarin or a direct oral anticoagulant. In light of the p.R42H variant and the patient's history of normal protein C activity levels on testing (131 IU dL−1 2 years prior to presentation, 86 IU dL−1 1 year prior), additional protein C testing was performed. Her protein C antigen was normal (78%, laboratory reference range: 70%-140%, age-specific published reference range: 40%-92%1) and activity via the amidolytic assay was similar to previous results (83 IU dL−1), but was <10 IU dL−1 via the clot-based assay (Functional Protein C, Diagnostica Stago, Inc.). Protein C and genetic testing were performed on the patient's parents, brother, and maternal half-sister (Figure 2). This testing demonstrated that her half-sister is a heterozygote for the maternally-inherited variant and that her brother does not have either variant PROC gene. Neither the patient's mother nor father has had thrombotic complications. There is, however, a significant history of venous thromboembolism (VTE) on the maternal side: maternal grandmother had a deep vein thrombosis in her 30s and two maternal aunts and one uncle had strokes in their 40s and 50s. There was no known history of VTE on the paternal side. Prior studies have identified families with protein C deficiency without known thrombotic episodes,5 as well as families where co-inherited modifying genetic factors (such as Factor V Leiden) affect thrombotic risk.6 As expected, given the presence of the p.R42H variant, her father had normal protein C activity by amidolytic method, with a clot-based assay activity approximately 50% lower. Protein C is a Vitamin K-dependent glycoprotein that plays a pivotal role in normal hemostasis by downregulating the coagulation cascade. After hepatic synthesis, protein C undergoes posttranslational modifications7 and is secreted as one light chain and one heavy chain connected by a single disulfide bond.8, 9 Activated protein C (APC) proteolytically inactivates procoagulant factors Va and VIIIa,10, 11 and protein S accelerates its anticoagulant function.12 Protein C activation occurs on vascular endothelial surfaces when the thrombin-thrombomodulin complex cleaves the activation peptide from the N-terminus of the heavy chain.8, 13 For optimal activation, protein C must also bind Ca2+ and endothelial protein C receptor (EPCR).14 Calcium binds in the C-terminal heavy chain of the molecule, which contains the APC catalytic domain15 and the essential activation peptide.8 The N-terminal light chain of APC is non-catalytic but binds both EPCR and protein S at a γ-carboxyglutamic (Gla) domain. After this multi-step process of activation, protein C acquires its enzymatic activity.8 Protein C deficiency is associated with an increased risk of thrombosis, with clots in a variety of anatomic locations having been described in individuals with the deficiency. Both homozygous deficiency and compound heterozygous variants have been associated with neonatal purpura fulminans. Heterozygous deficiency is associated with a less severe initial presentation, though there is an estimated 50% rate of thrombotic events by age 40 in heterozygotes.16 Interestingly, despite her compound variants, our patient developed purpura fulminans as a toddler, rather than as a neonate. On review of her record, she did have other common manifestations of homozygous protein C deficiency including multiple strokes and vision loss, presumably due to retinal artery thrombi. Protein C deficiency can be divided into two major categories: Type I quantitative deficiencies and Type II qualitative deficiencies. Type II deficiencies can be further subclassified into Type IIa - those that occur as a result of alterations to the catalytic or activation sites - and Type IIb - those that occur in sites with other protein functions (Table 1).17, 18 Diagnostic assays for protein C deficiency are either immunologic or functional.19, 20 Antigenic assays quantify protein C via binding of an antibody reagent to protein C in patient plasma. Antigenic assays measure the quantity of protein C antigen regardless of function, and therefore cannot detect Type II deficiencies. Type I deficiencies show a proportionate decrease in protein C antigen and activity independent of testing method. Functional assays are either clot-based or chromogenic (amidolytic). In both assays, protein C in patient plasma is first activated by a rapid activator such as Southern Copperhead (Agkistrodon contortrix contortrix) venom. Amidolytic assays measure the ability of APC to cleave a synthetic peptide substrate, releasing a detectable chromogenic compound. Therefore, amidolytic assays are limited because they only measure protein C activation and catalytic site function.17 In contrast, clot-based assays rely on the patient's APC to cleave activated coagulation factors Va and VIIIa, thereby prolonging either a PTT (prolonged by cleavage of FVa and FVIIIa) or Russell viper venom-based clotting time (prolonged by cleavage of only FVa).20 Some clot-based assay results can be affected by high levels of factor VII21; APC-resistant Factor V Leiden22; and coagulation inhibitors such as heparin or lupus anticoagulant23, 24 (for a summary, see Table 2). Type I Type IIA Type I Types IIA & IIB Type I Types IIA & IIB In families with Type II protein C deficiency, some kindreds will show a normal antigen and amidolytic assay result, but abnormal clot-based assay result.4, 17 This discrepancy is a consequence of the particular protein C domain affected by the individual's variant. Variants that affect substrate (FVa and FVIIIa) binding, protein S binding, or cell surface binding cause low clot-based protein C activity but normal antigen and amidolytic assay results. This patient's protein C activity was checked multiple times via the amidolytic assay and was always normal. After the results of whole exome sequencing returned, a blood sample was sent to ARUP Laboratories (Salt Lake City, Utah) where a clot-based protein C assay (Functional Protein C, Diagnostica Stago, Inc.) using electromagnetic mechanical clot detection and assessment of aPTT prolongation was performed and showed very low protein C activity (<10 IU dL−1). Our patient's paternally inherited variant is in the non-catalytic Gla domain, and therefore predicted to affect EPCR and protein S binding and result in discrepant amidolytic and clot-based assay results. The maternal variant results in the amino acid deletion of Gln 112 in the region of the second epidermal growth factor (EGF)-like domain of the light chain of APC.3 While the role of EGF-like domains in APC anticoagulant function is unknown, both our patient's clinical presentation and the variant's original description are consistent with impaired protein C anticoagulant activity that does not affect protease activity. In hindsight, our patient's underlying protein C deficiency explains her clinical improvement in response to plasmapheresis: the procedure replaced her circulating, defective protein C with functional protein C restoring appropriate regulation of her coagulation cascade. If the protein C deficiency had been recognized initially, the preferred therapy would have been infusion of a protein C concentrate as opposed to plasma; commercially available preparations exist and have been used in both the acute and prophylactic setting for individuals with severe protein C deficiency.25 Protein C from transfused plasma has a half-life of a few hours; in our patient, the volume and repeated plasma administration with plasmapheresis likely provided enough protein C to restore normal hemostasis. This case points to the importance of performing clot-based assays when high suspicion for protein C deficiency exists despite normal amidolytic assay results; the use of a physiologic substrate will identify defects in sites outside the catalytic domain of protein C and increase the likelihood of detecting a deficiency in functional protein. The authors declare no conflicts of interest with the content of this paper. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.