The presented data identified approximately 231 distinct proteins after the 14 most abundant proteins, albumin, etc., were removed by affinity chromatography. Of no surprise, female plasma had higher levels of pregnancy zone protein, which is directly related to the sex hormones responsible for gender differences. Surprisingly, females also had significantly more factor V and increased amounts of α1
-antitrypsin and β2
-microglobulin. These differences were confirmed by standard biochemical assays, including: factor V activity in a CAP, CLIA and FDA-certified clinical coagulation laboratory and western blotting, respectively. Males plasma showed an increase in Fc-binding protein, protein Z-dependent protease inhibitor, phosphatidylinositol-glycan specific phospholipase, protein S-100, and transgelin-2. One limitation, other than small sample size, is that this study includes data from only 5 A+ males and 3 A+ and 2 O+ females. However, none of the proteins that demonstrated numerical distinctions between male and female donors have been shown to differ among ABO or Rh blood groups, similar to factor VIII or von Willebrand factor, and there were no significant differences noted between the plasma from A+ or O+ females.4
In addition, the complement factors H and C4B, demonstrated a 1.5- to 2-fold difference in our analysis, the smallest fold difference of the proteins analyzed with a statistical difference between the spectral counts of p<.005.
Factor V is a vital coagulation co-factor that requires Ca2+
and a platelet phospholipid surface to accelerate the formation of factor Xa from hours to seconds, and allows for the productive recruitment of pro-thrombin to the platelet membrane for efficient thrombin generation. 4
In addition, free Factor Va markedly facilitates clearance of the Va:Xa complex to decrease widespread thrombin generation.4,17
Factor V levels increase with age 6-7.6% per decade of life.18,19
Deficiencies in Factor V results in minor to major bleeding and the treatment involves the infusion of frozen plasma; however, factor V is the most labile of the coagulation factors in frozen plasma such that one should transfuse plasma of 1-2 months storage age to treat deficiency.4,17,20
The presented proteomic data demonstrates that factor V procoagulant levels are higher in the donated plasma units from 5 females than 5 males despite the males being significantly older (Males: 59.8 years vs. females: 41 years, p=0.02). This difference was confirmed by a factor V activity assay employing factor V-deficient plasma. However, one would expect that factor V levels in male plasma, especially from significantly older males, would be greater or equal to factor V levels in the plasma from younger female donors, which was not the case. Furthermore, the factor V levels are from plasma units that were stored for identical times and not from freshly isolated plasma samples as was the case for previous studies that concluded that there is not a difference in factor V from males and females.18,19
Further studies with a large population of donors is required to corroborate these findings.
α-1-antitrypsin is the best known serine protease inhibitor of the serpin family which has a structural conformation that allows it to bind tightly and inhibit serine proteases, including: neutrophil elastase, cathepsin G, and proteinase 3.21-23
α-1-antitrypsin deficiency is manifested by protease-induced bronchiectasis, lung injury, and liver damage including: cholestasis, cirrhosis, hepatocellular carcinoma, and neutrophil cutaneous panniculitis. 21-23
α-1-antitrypsin inhibits neutrophil elastase both in vitro
and in vivo
and has been successful in treating the pulmonary but not the liver manifestations of α-1-antirypsin deficiency, and adults with cystic fibrosis in a small clinical study.21-23
From our previous experience with immunoaffinity depletion (ID) of plasma for MS analysis it was not surprising to observe peptides arising from proteins that the ID column was directed against. In general, we observe 85 to 98% depletion efficiency of the intact protein as long as the binding capacity of the column is not exceeded. While the antibodies used are usually polyclonal there is the possibility that cleavage or modifications could prevent epitope recognition. To validate the difference observed in α1-antitrypsin levels we performed Immunoblotting of unmodified (undepleted) plasma and found the aforementioned differences in female vs. male plasma.
-microglobulin, which is the light or β-chain of the HLA class I molecule, is present in serum, urine, and synovial fluid, and because of its purely renal excretion has been used as a marker of renal function.24,25
-microglobulin is increased in the serum or plasma of patients with chronic kidney disease, hematological malignancies, auto-immune disease, especially systemic lupus erythematosis, infections and in lymphoproliferative disorders, e.g. myeloma; however, little data exists about plasma levels in healthy adults.24-30
In the sera, adolescent males are reported to have higher levels of β2
-microglobulin than females; however, β2
-microglobulin levels in the sera from adults demonstrate no increase in males and a mild increase with age.29,31
To the best of our knowledge there are no reports of relative β2-microglobulin plasma levels in males versus females.
Increases in the complement proteins C4 and factor H have not been linked to clinical disease; however deficiencies of C4 result in immune complex disease and deficiencies of factor H have been linked to recurrent pyogenic infections.32
C4 is the substrate for C1 to activate the classical pathway of complement activation and Factor H, along with Factor I, controls activation of the alternative pathway.32
There are no data with regard to gender differences in the complement proteins.
In FDA-licensed plasma from male donors a number of proteins were increased vs. the proteins from female plasma. Protein S-100 is a protein expressed by neural glia in the central nervous system and is used as a marker for brain tumors and other malignancies and lymphoproliferative diseases.33,34
The function of S-100 proteins are multifaceted and have not been clearly defined.33,34
Protein Z-dependent protease inhibitor (ZPI) is a member of the serpin superfamily of protease inhibitors which inhibits both Factor Xa and Factor Xia activity with the former requiring protein Z binding factor Xa in a Ca2+
ZPI is consumed during coagulation; however, it forms weak complexes with factor Xa and Xia and serves as a co-factor with protein Z to dampen coagulation.35,36
Fc-binding protein is widely expressed in the tissues of human and on the epithelial surface, especially the mucosal epithelium, and it is functionally intact in mucus, saliva, and other fluids and appears to be an important part of mucosal membrane defenses.37-39
In addition, Fc-binding proteins can inhibit complement mediated interactions and help protect the host from foreign immunoglobulins.37-39
Transgelin-2 is a structural protein from smooth muscle that has been reported to be a marker of lung adenocarcinoma.40,41
Furthermore, transgelin-2 appears to be synthesized by B-1 and not B-2 lymphocytes from the spleen.40
. The function of transgelin-2 remains unknown, and although it is reported to be a structural protein of smooth muscle, its presence in epithelial stroma, B-1 lymphocytes, and pulmonary adenocarcinoma remain undefined.40,41
Lastly, phosphatidylinositol-glycan specific phospholipase is an intracellular phospholipase responsible for the generation of second messengers from phosphatidylinositol 4,5-bisphosphate (PIP2
) most notably inositol 1,4,5-trisphosphate (IP3
) and inositol 1,3,4,5-tetrakisphosphate (IP4
Its significance in male plasma is entirely unknown but it appeared in all 5 plasma samples and was significantly increased in the plasma units from male versus female donors.
Recent data has demonstrated differences between patients with solid tumors and healthy controls to some of the more abundant proteins removed by affinity depletion in these studies, namely α-1-antitrypsin, transthyretin, and apolipoprotein AI.45
Follow-up studies demonstrated that both transthyretin and apolipoprotein AI (both decreased) used in combination with the tumor marker CA125 were reliable predictors of early-stage ovarian cancer; however none of these proteins alone were sensitive or specific enough to predict ovarian cancer. Follow-up data has not demonstrated the specificity or the sensitivity of the earlier studies.46-48
In addition, both transthyretin and α-1-antitrypsin have been implicated as sensitive and specific tumor markers of pancreatic cancer; conversely, newer data has demonstrated that when the studies accounted for obstructive jaundice, only transthyretin remained as a sensitive and specific tumor marker.45,49,50
Further work is needed to ascertain the best tumor markers for specific age groups, it is notable that differences in α-1-antitrypsin via gender in healthy adult blood donors were found in the presented data set.
Our data indicate that despite the small sample size used for discovery there are protein differences in FDA-licensed plasma that reflect donor gender. Several differences were corroborated by standard biochemical quantification: factor V, β2-microglobulin, and α-1-antitrypsin. It is possible that our observed differences are artifacts due to the small sample size, and studies with larger populations need to be completed to validate these data. The roles of proteins found at higher abundance in either male or female plasma remain undefined; nevertheless, these results were not foreseen. Plasma infusions are important to replace coagulation factors, especially factors II, V, and VII and a case may be made for factor X in injured patients. In addition, the role of proteases in acute organ injury and multiple organ failure have been suggested, and if correct, female plasma could more efficiently be used as part of resuscitation to augment factor V levels, to increase circulating anti-protease concentrations, and to potentially decrease indiscriminant alternative pathway activation of complement. More boldly stated, this study suggests that there are “trade-offs” with every policy including the transfusion of male-only plasma such that while a specific policy may help increase blood safety with some patients, e.g. TRALI, it may reduce the optimal transfusion benefits for other patient types. Clearly, though, these advantages are theoretical and would require careful outcome analyses in well-matched patient cohorts before any definitive statement could be made.